Purine-derived substance-producing bacterium and a method for producing purine-derived substance

ABSTRACT

A purine-derived substance is produced by culturing a  Bacillus  bacterium which has an ability to produce a purine-derived substance and has enhanced activity of an enzyme of the oxidative pentosephosphate pathway. The purine-derived substance is produced in the medium or the bacterial cells, and can be collected from the medium or the bacterial cells.

The present application claims priority under 35 U.S.C. § 119(a) toJapanese Patent Application Nos. 2005-067560, filed Mar. 10, 2005, and2005-280186, filed Sep. 27, 2006, the entireties of which areincorporated by reference. Also, the Sequence Listing on compact diskfiled herewith is hereby incorporated by reference (File name: US-271Seq List; File size: 59 KB; Date recorded: Mar. 9, 2006).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a Bacillus bacterium which is usefulfor the production of purine-derived substances, including purinenucleotides such as 5′-inosinic acid and 5′-guanylic acid, and purinenucleosides such as inosine and guanosine. These nucleosides areimportant as starting materials for making purine nucleotides.Purine-derived substances are useful as seasonings, pharmaceuticals, andraw materials thereof.

2. Brief Description of the Related Art

Methods for producing inosine and guanosine by fermentation usingadenine-auxotrophic mutants of Bacillus bacteria and derivatives thereofwhich are imparted with resistance to various drugs such as purineanalogs (JP38-23099B, JP54-17033B, JP55-2956B, JP55-45199B, JP57-14160B,JP57-41915B, JP59-42895A, and US2004-0166575 A) have been previouslydescribed. Also, methods using mutants of Brevibacterium bacteria(JP51-5075B, JP58-17592B and Agric. Biol. Chem., 1978, 42, 399-405) havebeen described.

Such mutants are typically obtained by treating the cells with UVirradiation or N-methyl-N′-nitro-N-nitrosoguanidine, and selecting atarget mutant in a suitable selective medium.

Strains which produce purine-derived substances have also been bredusing genetic engineering techniques in Bacillus bacteria (JP58-158197A,JP58-175493A, JP59-28470A, JP60-156388A, JP1-27477A, JP1-174385A,JP3-58787A, JP3-164185A, JP5-84067A, and JP5-192164A), Brevibacteriumbacteria (JP63-248394A), and Escherichia bacteria (WO99/03988).Specifically, a method of efficiently producing nucleic acid-derivedsubstances such as hypoxanthine, uracil, guanine, and adenine with aBacillus bacterium in which a gene (purR gene) encoding the purineoperon repressor is disrupted is disclosed in U.S. Pat. No. 6,284,495.

In Bacillus subtilis, the purine operon repressor is known to regulatethe expression of the purA. This gene is involved in AMP biosynthesis(Proc. Natl. Acad. Sci, USA, 1995, 92, 7455-7459). This repressor alsoregulates the expression of the glyA gene, which is involvedformyltetrahydrofolate biosynthesis (J. Bacteriol., 2001, 183,6175-6183), and the pbuG gene which encodes a hypoxanthine/guaninetransporter (J. Bacteriol., 2003, 185, 5200-5209.), in addition to thepurine operon gene.

A method for breeding a strain which efficiently produces inosine bydisrupting the succinyl-AMP-synthase gene (purA) to impartadenine-auxotrophy and disrupting the purine nucleoside phosphorylasegene (deoD) to inhibit the decomposition of inosine into hypoxanthine inaddition to disrupting the purR gene is described in US2004-0166575 A.

Meanwhile, in the oxidative pentosephosphate pathway, glucose isphosphorylated by glucose kinase to generate glucose-6-phosphate, whichis oxidatively converted to ribose-5-phosphate. However, therelationship between this pathway and the biosynthetic pathway ofpurine-derived substances is not well understood; and therefore, it wasnot expected that a bacterium which produces purine-derived substancescould be obtained by modifying the oxidative pentosephosphate pathway ofthe bacterium.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a bacterium suitablefor producing purine-derived substances such as purine nucleosides andpurine nucleotides by fermentation, and to provide a method forproducing purine-derived substances using such a bacterium.

The inventors of the present invention conducted extensive studies forsolving the above-mentioned object, and found that the ability toproduce purine-derived substances such as purine nucleosides and purinenucleotides of a Bacillus bacterium could be enhanced by increasing anactivity of an enzyme of the oxidative pentosephosphate pathway,particularly the activity of glucose-6-phosphate dehydrogenase orribose-5-phosphate isomerase. They also found that the ability toproduce a purine-derived substance in a Bacillus bacterium could befurther enhanced by further modification which results in enhancing theexpression of a gene encoding phosphoribosylpyrophosphate (PRPP)synthetase or a gene encoding an enzyme involved in purine nucleotidebiosynthesis, or to decrease the activity of purine nucleosidephosphorylase. Based on these findings, the present invention has beencompleted.

It is an object of the present invention to provide a Bacillus bacteriumhaving an ability to produce a purine-derived substance, wherein thebacterium is modified to enhance an activity of an enzyme of theoxidative pentosephosphate pathway.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said purine-derived substance isselected from the group consisting of inosine, xanthosine, guanosine,and adenosine.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said purine-derived substance isselected from the group consisting of inosinic acid, xanthylic acid,guanylic acid, and adenylic acid.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said enzyme isglucose-6-phosphate-dehydrogenase or ribose-5-phosphate isomerase.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said activity of the enzyme isenhanced by increasing the copy number of a gene encoding the enzyme ormodifying an expression control sequence of the gene.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said enzyme is glucose-6-phosphatedehydrogenase which is selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 48; and

(B) a protein comprising the amino acid sequence of SEQ ID NO: 48,wherein one or several amino acids are substituted, deleted, inserted,added, or inverted, and said protein has glucose-6-phosphatedehydrogenase activity.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said enzyme is ribose-5-phosphateisomerase which is selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 50; and

(B) a protein comprising the amino acid sequence of SEQ ID NO: 50wherein one or several amino acids are substituted, deleted, inserted,added, or inverted, and said protein has ribose-5-phosphate isomeraseactivity.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein 1-20 amino acids are substituted,deleted, inserted, added, or inverted.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said enzyme is glucose-6-phosphatedehydrogenase and the gene encoding said enzyme is selected from thegroup consisting of:

(A) a DNA comprising the nucleotide sequence of SEQ ID NO: 47; and

(B) a DNA that is able to hybridize with a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 47, or a probeprepared from the nucleotide sequence under stringent conditions, andencodes a protein having glucose-6-phosphate dehydrogenase activity.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein said enzyme is ribose-5-phosphateisomerase and the gene encoding said enzyme is selected from the groupconsisting of:

(A) a DNA comprising the nucleotide sequence of SEQ ID NO: 49; and

(B) a DNA that is able to hybridize with a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 49, or a probeprepared from the nucleotide sequence under stringent conditions, andencodes a protein having ribose-5-phosphate isomerase activity.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein the bacterium is further modifiedto enhance phosphoribosylpyrophosphate synthetase activity.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein the bacterium is further modifiedto enhance the expression of purine operon.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein the expression of the purineoperon is enhanced by disrupting a purR gene that encodes a purineoperon repressor or deleting a portion of an attenuator region of thepurine operon.

It is a further object of the present invention to provide the Bacillusbacterium as described above, wherein the bacterium is further modifiedto reduce the activity of purine nucleoside phosphorylase.

It is a further object of the present invention to provide a method forproducing a purine-derived substance comprising:

culturing the Bacillus bacterium as described above in a medium; and

collecting said purine-derived substance.

It is a further object of the present invention to provide the method asdescribed above, wherein said purine-derived substance is a purinenucleoside or purine nucleotide.

It is a further object of the present invention to provide the method asdescribed above, wherein said purine-derived substance is selected fromthe group consisting of inosine, xanthosine, guanosine, and adenosine.

It is a further object of the present invention to provide the method asdescribed above, wherein said purine-derived substance is selected fromthe group consisting of inosinic acid, xanthylic acid, guanylic acid,and adenylic acid.

It is a further object of the present invention to provide a method forproducing a purine nucleotide comprising:

producing a purine nucleoside by the method as described above;

reacting the purine nucleoside with a microorganism which has an abilityto produce a nucleoside-5′-phosphate ester, or with an acid phosphatase,in the presence of a phosphate donor selected from the group consistingof phosphoric acid, phenyl phosphate, and carbamyl phosphate to producepurine nucleotide; and

collecting the purine nucleotide.

The Bacillus bacterium of the present invention can be used toefficiently produce purine-derived substances such as purine nucleosidesand purine nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of upstream of the purine operon. Thenucleotide sequence enclosed in boxes denotes the purine operonpromoter; the overlined sequence denotes an antiterminator dyad, and theunderlined sequence denotes a terminator dyad. The sequence which willbe deleted (75 bp) is indicated by upper-case letters.

FIG. 2 shows the transcription activity of the modified purine operonpromoter.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

<1>Bacillus Bacterium of the Present Invention

(I) Imparting the Ability to Produce a Purine-Derived Substance

The Bacillus bacterium of the present invention has an ability toproduce a purine-derived substance and has been modified to enhance theactivity of an enzyme of the oxidative pentosephosphate pathway.

The term “purine-derived substance” means a substance having a purineskeleton, and examples thereof include purine nucleosides and purinenucleotides. Examples of purine nucleosides include inosine, xanthosine,guanosine, and adenosine. Examples of purine nucleotides include5′-phosphate ester of purine nucleosides, specifically, inosinic acid(inosine 5′-monophosphate, also referred to as “IMP” hereinafter),xanthylic acid (xanthosine 5′-monophosphate, also referred to as “XMP”hereinafter), guanylic acid (guanosine 5′-monophosphate, also referredto as “GMP” hereinafter), and adenylic acid (adenosine 5′-monophosphate,also referred to as “AMP” hereinafter).

The phrase “ability to produce a purine-derived substance” means thatthe Bacillus bacterium of the present invention has an ability toproduce and cause accumulation of a purine-derived substance in a mediumor in the bacterial cells to such an extent that the substance can becollected from the medium or the bacterial cells when it is cultured inthe medium. The Bacillus bacterium of the present invention may have anability to produce two or more purine-derived substances.

The Bacillus bacterium of the present invention may originally possessthe ability to produce a purine-derived substance, or this ability maybe imparted by modifying a Bacillus bacterium such as those shown belowby a mutagenesis or gene recombination techniques. Furthermore, theBacillus bacterium of the present invention may have the abilityimparted by a modification which enhances the activity of an enzyme ofthe oxidative pentosephosphate synthetic pathway.

Examples of the parent strain which can be used to obtain the Bacillusbacterium of the present invention include Bacillus subtilis, Bacillusamyloliquefaciens, and Bacillus pumilus. Examples of Bacillus subtilisinclude Bacillus subtilis 168 Marburg strain (ATCC6051) and Bacillussubtilis PY79 strain (Plasmid, 1984, 12, 1-9). Examples of Bacillusamyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

The Bacillus bacterium having an ability to produce inosine can beobtained by imparting an adenine-auxotrophy, as well as resistance to adrug such as a purine analog to the Bacillus bacterium as describedabove (JP38-23099B, JP54-17033B, JP55-45199B, JP57-14160B, JP57-41915B,and JP59-42895B, US2004-0166575A). A mutant of Bacillus bacterium havingsuch an auxotrophy and drug-resistance can be obtained by treating thebacterium with a mutagenesis agent which is commonly employed tointroduce mutations, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG)and ethane methane sulfonate (EMS).

An example of an inosine-producing strain of Bacillus bacterium includesthe Bacillus subtilis KMBS16 strain. This strain is a derived fromBacillus subtilis trpC2 strain (168 Marburg), wherein a purR geneencoding a purine operon repressor, a purA gene encoding succinyl-AMPsynthase, and a deoD gene encoding purine nucleoside phosphorylase aredisrupted (purR::spc, purA::erm, deoD::kan) (US2004-0166575A). Bacillussubtilis AJ3772 strain (FERM P-2555) (JP62-014794A) Bacillus subtilis6-ethoxypurine resistant strain (US2004-0166575A) may also be used.

Examples of a Bacillus bacterium which has an ability to produceguanosine include a Bacillus bacterium which has enhanced IMPdehydrogenase activity (JP3-58787A) and a Bacillus bacterium which isobtained by introducing a vector comprising a gene conferring resistantto a purine analog and decoyinine into an adenine-auxotrophic mutant(JP4-28357A).

Examples of a Bacillus bacterium which has an ability to produce apurine nucleotide include inosine-producing strains of Bacillus subtiliswhich have attenuated phosphatase activity (Uchida, K. et al, Agr. Biol.Chem., 1961, 25, 804-805; Fujimoto, M., Uchida, K., Agr. Biol. Chem.,1965, 29, 249-259), and mutant of a Bacillus bacterium which has anability to produce 5′-guanylic acid, imparted with anadenine-auxotrophy, and resistance to decoyinine or methionine sulfoxide(JP56-12438B).

Examples of a method for breeding a Bacillus bacterium which has theability to produce a purine-derived substance include enhancing theactivities of enzymes involved in purine biosynthesis, and which arecommon to the biosynthesis of purine nucleosides and nucleotides. Theactivity of the enzymes is preferably enhanced to a level greater thanthat of unmodified strain of Bacillus bacterium, such as a wild-typestrain of Bacillus bacterium. The phrase “activity is enhanced”encompasses when the number of enzyme molecules per cell is increased,and when the relative activity of the enzyme molecule is increased. Forexample, the activity can be enhanced by increasing the expression ofthe gene encoding the enzyme.

Examples of an enzyme involved in the biosynthesis of purine includephosphoribosylpyrophosphate (PRPP) amidotransferase and PRPP synthetase.

Some of the catabolites derived from sugar sources such as glucose thatflow into the pentosephosphate system are converted toribose-5-phosphate via ribulose-5-phosphate. PRPP, which is aindispensable precursor in the biosynthesis of purine nucleosides,histidine, and tryptophan, is produced from ribose-5-phosphate.Specifically, the ribose-5-phosphate is converted to PRPP by PRPPsynthetase [EC: 2.7.6.1]. Accordingly, modifying a Bacillus bacterium toenhance the activity of PRPP synthetase imparts the ability to produce apurine-derived substance to the Bacillus bacterium, and the combinedenhancement of the PRPP synthetase activity with an activity of anenzyme involved in the pentose-phosphate system is more effective.

The phrase “the activity of PRPP synthetase is enhanced” means that theactivity of PRPP synthetase increases as compared to an unmodifiedstrain, such as a wild-type strain or a parent strain. The activity ofthe PRPP synthetase can be measured by the method of Switzer et al.(Methods Enzymol., 1978, 51, 3-11)) or Roth et al. (Methods Enzymol.,1978, 51, 12-17). A Bacillus bacterium in which the activity of the PRPPsynthetase is enhanced can be obtained by increasing the expression of agene encoding the PRPP synthetase in the same manner as described inUS2004-0166575A, for example, by using a plasmid or integrating the geneinto a chromosome. An example of a gene which encodes the PRPPsynthetase includes the prs gene (SEQ ID NO: 57, GenBank Accession No.X16518) derived from a Bacillus bacterium; however, any gene encoding aprotein having PRPP synthetase activity in a Bacillus bacterium,including genes derived from other bacteria and genes derived fromplants and animals, can also be used.

When PRPP is produced, some of it is converted to purine nucleosides andpurine nucleotides by the enzymes involved in purine biosynthesis. Theenzymes involved in purine biosynthesis are encoded by the purineoperon, and examples of the purine operon include thepurEKB-purC(orf)QLF-purMNH(J)-purD operon gene from Bacillus subtilis(Ebbole D J and Zalkin H, J. Biol. Chem., 1987, 262, 17, 8274-87) (alsoknown as purEKBCSQLFMNHD: Bacillus subtilis and Its Closest Relatives,Editor in Chief: A. L. Sonenshein, ASM Press, Washington, D.C., 2002)and the genes of the pur regulon from Escherichia coli (Escherichia andSalmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM press,Washington D.C., 1996).

Accordingly, enhancing the expression of these genes imparts the abilityto produce a purine-derived substance. However, genes of the purineoperon used in the present invention are not limited to these genes, andgenes derived from other microorganisms and from plants and animals mayalso be used.

Expression of the genes of the pur operon can be enhanced in a Bacillusbacterium by using a plasmid containing the gene or integrating the geneinto a chromosome in the same manner as enhancing the gene encoding theenzyme of the oxidative pentosephosphate pathway as described below.

The expression of the purine operon can also be enhanced by replacing apromoter of the purine operon with a stronger one, or by replacing the“−35 region”, or “−10 region” of the native promoter with a consensussequence.

For example, in Bacillus subtilis (B. subtilis 168 Marburg strain;ATCC6051), the “−35 region” of the purine operon is a consensus sequence(TTGACA), but the “−10 region” is TAAGAT, which differs from theconsensus sequence TATAAT (Ebbole, D. J. and H. Zalikn, J. Biol. Chem.,1987, 262, 8274-8287). Accordingly, by changing the “−10 sequence”(TAAGAT) to the similar consensus sequence TATAAT, TATGAT, or TAAAAT, itis possible to enhance the transcription activity of the purine operon.A promoter sequence can be replaced by gene substitution, which isdescribed below.

The expression of the purine operon can also be enhanced by decreasingthe expression of a purine operon repressor (U.S. Pat. No. 6,284,495).

To decrease the expression of the purine operon repressor, for example,a Bacillus bacterium is treated with a mutagenesis agent commonly usedin mutation treatment, such as UV irradiation, NTG, or EMS, and themutants having reduced expression of the purine operon repressor areselected.

Furthermore, the expression of the repressor may be decreased byperforming homologous recombination (Experiments in Molecular Genetics,Cold Spring Harbor Laboratory Press (1972); Matsuyama, S. and Mizushima,S., J. Bacteriol., 1985, 162, 1196-1202), so that a gene encoding apurine operon repressor (purR) on the chromosome (GenBank Accession No.NC_(—)000964, SEQ ID NO: 51) is replaced with a mutant gene in whichpart of the sequence of the repressor is deleted (hereinafter, this maybe referred to as a “disrupted-type gene”).

For example, a wild-type gene can be replaced with a disrupted-type geneon the host chromosome in the manner as described below. Hereinafter,the disruption of the purR gene is explained. Other genes such as thepurA gene and deoD gene can be similarly disrupted.

In homologous recombination, a plasmid that is not capable ofreplicating in cells of a Bacillus bacterium and which has a nucleotidesequence that is homologous with a sequence on the chromosome isintroduced into the bacterial cell, resulting in homologousrecombination. The plasmid is then recombined into the chromosome in thefirst recombination (single-cross-over recombination). Then, a secondrecombination (double-cross-over recombination) occurs and the plasmidis removed from the chromosome. At this time, in some cases, adisrupted-type gene on the plasmid is integrated into the chromosome andthe wild-type gene on the chromosome is removed with the plasmid portionfrom the chromosome. By selecting such bacterial strains, it is possibleto obtain bacterial strains in which the wild-type purR gene on thechromosome has been replaced with the disrupted-type purR gene.

Such techniques are established and include a method of using a linearDNA, a method using a temperature-sensitive plasmid, and the like.Furthermore, the purR gene may be disrupted by using a plasmid in whicha drug-resistance marker gene has been inserted into the native purRgene which prohibits replication in the target bacterial cell. That is,in a bacterial cell that has been transformed with this plasmid, themarker gene is incorporated into the chromosomal DNA and imparts drugresistance. Since the marker gene is integrated into the chromosome at ahigh rate by homologous recombination of the purR gene sequences thatsandwiches the marker gene on the plasmid with the wild-type purR geneon the chromosome, a purR gene-disrupted strain can be efficientlyselected.

Specifically, the disrupted-type purR gene used in gene disruption canbe obtained by digesting a wild-type purR gene with restriction enzymesto delete a certain region of the purR gene, followed by self-ligationof the digested DNA, or inserting another DNA fragment (marker geneetc.) into the wild-type purR gene, or causing replacement, deletion,insertion, addition, or inversion of one or several nucleotides in thecoding region or promoter region of the nucleotide sequence of the purRgene by a site-specific mutation method (Kramer, W. and Fritz, H. J.,Methods Enzymol., 1987, 154, 350-367) or by recombinant PCR (PCRTechnology, Stockton Press (1989)) or by treatment with a chemical agentsuch as sodium sulfite or hydroxylamine (Shortle, D. and Nathans, D.,Proc. Natl. Acad. Sci. USA, 1978, 75, 2170-2174), followed by selectionof a strain in which the activity of the purine operon repressor isdecreased or eliminated or a strain in which transcription of the purRgene is decreased or eliminated. Of these methods, deleting a certainregion of the wild-type purR gene by digestion with restriction enzymesfollowed by self-ligation and inserting another DNA fragment into thewild-type purR gene are preferable in view of reliability and stability.

The purR gene can be obtained by PCR using oligonucleotides preparedbased on the nucleotide sequence of the known purR gene as primers, andthe chromosomal DNA from a microorganism having a purine operon, or thelike as a template. Furthermore, the purR gene can also be obtained byhybridization using an oligonucleotide probe based on the nucleotidesequence of the known purR gene of a chromosomal DNA library ofmicroorganisms having purine operon, or the like. The nucleotidesequence of the purR gene of the Bacillus subtilis 168 Marburg strainhas been reported (GenBank Accession No. D26185 (the coding region isthe nucleotide numbers 118041-118898) and NC_(—)000964 (the codingregion is the nucleotide numbers 54439-55296)). The nucleotide sequenceof the purR gene and the amino acid sequence encoded by the gene areshown in SEQ ID NOS: 51 and 52 (also disclosed in US2004-0166575A).

Primers for cloning the purR gene are not particularly limited as longas they can function in PCR to amplify the purR gene, and specificexamples thereof include oligonucleotides having the nucleotidesequences of SEQ ID NO: 59 (GAAGTTGATGATCAAAA) and SEQ ID NO: 60(ACATATTGTTGACGATAAT).

A purR gene which can be used to prepare a disrupted-type purR gene doesnot necessarily contain the full length purR gene; a fragment of thepurR gene having a length sufficient to disrupt the purR gene may alsobe used. Furthermore, a bacterium which can be used to obtain the genefor preparing a disrupted-type purR gene is not particularly limited solong as it has a gene which is sufficiently homologous to causehomologous recombination with the purR gene on the chromosome of theBacillus bacterium. However, it is preferable to employ a gene derivedfrom a microorganism which is identical to the targeted Bacillusbacterium.

The DNA which is capable of inducing homologous recombination with thepurR gene on the chromosome of the Bacillus bacterium may be a DNAencoding a protein having an amino acid sequence of SEQ ID NO: 52,wherein one or several, for example, 1 to 50, preferably 1 to 30, andmore preferably 1 to 10 amino acids are substituted, deleted, insertedor added.

A DNA which is capable of inducing homologous recombination with thepurR gene on the chromosome of the Bacillus bacterium may also be a DNAhaving homology of not less than 70%, preferably not less than 80%, morepreferably not less than 90%, and still more preferably not less than95% to the nucleotide sequence of SEQ ID NO: 51. Such a DNA may also bea DNA that is able to hybridize under stringent conditions with the DNAhaving the nucleotide sequence of SEQ ID NO: 51. An example of stringentconditions includes washing at 1×SSC and 0.1% SDS, preferably 0.1×SSCand 0.1% SDS at 60° C.

Examples of a marker gene include drug-resistance genes such as thespectinomycin-resistance gene and kanamycin-resistance gene. Aspectinomycin-resistance gene derived from Enterococcus faecalis can beobtained by preparing plasmid pDG1726 from Escherichia coli ECE101strain, which is commercially available from the Bacillus Gentech StockCenter (BGSC), and removing a cassette portion from the plasmid. Anerythromycin-resistance gene of Staphylococcus aureus can be obtained bypreparing plasmid pDG646 from Escherichia coli ECE91 strain,commercially available from BGSC, and removing a cassette portion fromthe plasmid. Furthermore, a kanamycin-resistance gene derived fromStreptococcus faecalis can be obtained by preparing plasmid pDG783 fromEscherichia coli ECE94 strain and removing a cassette portion from theplasmid.

When a drug-resistance gene is used as the marker gene, a purRgene-disrupted strain can be obtained by inserting the drug-resistancegene at a suitable site in the purR gene on the plasmid, transforming abacterium with the resulting plasmid, and selecting transformants whichshow drug resistance. Disruption of the purR gene on the chromosome canbe confirmed by analyzing the purR gene on the chromosome or a markergene by Southern blotting or PCR. Incorporation of the above-describedspectinomycin-resistance gene, erythromycin-resistance gene, orkanamycin-resistance gene into the chromosomal DNA can be confirmed byPCR using primers capable of amplifying these genes.

Expression of the purine operon is known to be controlled by aterminator-antiterminator sequence (hereinafter, referred to as an“attenuator sequence”) positioned downstream of the promoter (Ebbole, D.J. and Zalkin, H., J. Biol. Chem., 1987, 262, 8274-8287; Ebbole, D. J.and Zalkin, H., J. Biol. Chem., 1988, 263, 10894-10902; Ebbole, D. J.and Zalkin, H., J. Bacteriol., 1989, 171, 2136-2141) (see FIG. 1).Accordingly, the expression of the purine operon can be enhanced bydeleting the attenuator sequence. The attenuator sequence can be deletedby the same method used for disrupting the purR gene.

To further increase the transcription of the purine operon, theabove-described methods may be combined. For example, the purine operonfrom which the attenuator sequence has been deleted may be amplifiedwith a plasmid, or multiple copies of such a purine operon may beintroduced into the chromosome, in a strain in which the purR gene isdisrupted.

Enhancing the activity of an enzyme involved in purine biosynthesis mayalso be achieved by eliminating the regulation of such an enzyme, forexample, by eliminating feedback inhibition of such an enzyme (WO99/03988).

The ability to produce a purine-derived substance may also be enhancedby attenuating the uptake of purine-derived substances by the cell. Forexample, the uptake of purine nucleosides by the cell may be attenuatedby blocking a reaction involved in the uptake of purine nucleosides bythe cell. An example of a reaction involved in the uptake of purinenucleosides includes reactions catalyzed by nucleoside permeases.

Furthermore, the ability to produce a purine-derived substance may alsobe enhanced by decreasing an activity of an enzyme involved indegradation of purine-derived substances. An example of such an enzymeincludes purine nucleoside phosphorylase.

Purine nucleotides biosynthesized from PRPP by the enzymes involved inpurine biosynthesis are dephosphorylated to purine nucleosides. Toefficiently produce purine nucleosides, it is preferable to decrease anactivity of purine nucleoside phosphorylases that degrade purinenucleosides into hypoxanthine and the like. That is, it is preferable todecrease or eliminate an activity of a purine nucleoside phosphorylasethat employs purine nucleosides, such as inosine, as substrates.

Specifically, this can be achieved by disrupting the deoD gene and pupGgene encoding purine nucleoside phosphorylase in a Bacillus bacterium.The Bacillus bacterium of the present invention may be modified byseparately or simultaneously disrupting the deoD gene and pupG gene. ThedeoD gene and pupG gene from Bacillus bacterium (deoD: GenBank AccessionNo. NC_(—)000964 (SEQ ID NO: 55), pupG: GenBank Accession No.NC_(—)000964 (SEQ ID NO. 53)) may be employed, and disruption of thesegenes can be performed in the same way as the disruption of the purRgene as described above.

The ability to produce a purine-derived substance may also be enhancedby decreasing an activity of inosine monophosphate (IMP) dehydrogenase.An example of a gene encoding IMP dehydrogenase includes a guaB gene. Anexample of the guaB gene includes the gene having the nucleotidesequence registered as Accession No. NC_(—)000964(15913 . . . 17376)inGenBank (SEQ ID NO: 61).

The ability to produce a purine-derived substance may also be enhancedby amplifying a gene encoding a protein having an activity to excretepurine-derived substances. An example of a bacterium in which such agene has been amplified includes a Bacillus bacterium in which the rhtAhomolog gene has been amplified (JP2003-219876A).

The microorganism used in the present invention may be modified toproduce a nucleoside or nucleotide by disrupting a gene encoding thecorresponding nucleosidase or nucleotidase. The precursors and theirrelated substances in the biosynthetic system of nucleoside ornucleotide may be produced by imparting an auxotrophy for inosine to thebacterium.

(II) Modification to Enhance an Activity of an Enzyme of the OxidativePentosephosphate Pathway.

The Bacillus bacterium of the present invention can be obtained bymodifying a bacterium having the ability to produce a purine-derivedsubstance as described above to enhance an activity of an enzyme of theoxidative pentosephosphate pathway. The modification to impart theability to produce a purine-derived substance and the modification toenhance the activity of an enzyme of oxidative pentosephosphate pathwaymay be performed in any order.

Herein, the term “oxidative pentosephosphate pathway” means the pathwayin which glucose that has been taken into the cell is phosphorylated byglucose kinase to glucose-6-phosphate, and glucose-6-phosphate isoxidatively converted to ribose-5-phosphate. Specific examples of anenzymes of oxidative pentosephosphate pathway includeglucose-6-phosphate dehydrogenase (EC: 1.1.1.49), 6-phosphate-gluconatedehydrogenase (EC: 1.1.1.44), and ribose-5-phosphate isomerase (EC:5.3.1.6). Among these enzymes, it is preferable that the activity of oneor both of glucose-6-phosphate dehydrogenase and ribose-5-phosphateisomerase is enhanced in the Bacillus bacterium of the presentinvention.

It is preferable that the activity of an enzyme of oxidativepentosephosphate pathway is enhanced as compared to a wild-type strainor an unmodified strain. The increase in an activity of such an enzymecan be measured by the following methods. For example, the enzymaticactivity of glucose-6-phosphate dehydrogenase can be measured bymeasuring the production of NADPH as described in (1) of Example 6, andthe enzymatic activity of ribose-5-phosphate isomerase can be measuredby measuring the production of ribulose-5-phosphate as described in (2)of Example 6.

The activity of the enzyme of the oxidative pentosephosphate pathway canbe enhanced by amplifying a gene encoding such an enzyme. The genes tobe amplified are not specifically limited as long as they encode aprotein having an activity of an enzyme involved in the oxidativepentosephosphate pathway. For example, genes derived from Bacillusbacterium may be employed.

An example of a gene encoding glucose-6-phosphate dehydrogenase includesa gene encoding the glucose-6-phosphate dehydrogenase of Bacillussubtilis which has an amino acid sequence of SEQ ID NO: 48, andpreferably includes a gene having the nucleotide sequence of SEQ ID NO:47 (the zwf gene: GenBank Accession No. NC_(—)000964). The zwf gene ispresent at 212 degrees on the chromosome of Bacillus subtilis.

An example of ribose-5-phosphate isomerase includes a gene encoding aprotein having amino acid sequence of SEQ ID NO: 50, and preferablyincludes a gene having a nucleotide sequence of SEQ ID NO: 49 (the ywlFgene: GenBank Accession No. NC_(—)000964). The ywlF gene is present at324 degrees on the chromosome of Bacillus subtilis in the vicinity ofthe glyA gene that encodes serine hydroxymethyl transferase.Ribose-5-phosphate isomerases include the enzyme known asribose-5-phosphate epimerase.

Genes encoding an enzyme of the oxidative pentosephosphate pathway maybe derived from a bacterium other than Bacillus bacterium, and may alsobe derived from plants or animals. A gene whose nucleotide sequence isalready known, and a gene obtained by isolating a gene encoding aprotein having an activity of an enzyme involved in the oxidativepentosephosphate pathway based on homology with the known nucleotidesequence from the chromosome of microorganisms, plants, and animals, andfollowed by sequence determination may be employed. A gene synthesizedbased on the nucleotide sequence may also be employed. Such genes may beobtained by amplifying a region containing a promoter and ORF byhybridization or PCR. Sequence information can be obtained from a publicdatabase such as GenBank.

In a Bacillus bacterium, the intracellular activity of an enzyme can beenhanced by increasing the expression of the gene encoding the enzyme.The expression of the gene can be increased by increasing the copynumber of the gene. For example, a fragment of a gene encoding theenzyme can be ligated to a vector that functions in a Bacillus bacteria,preferably a multi-copy vector, to prepare a recombinant DNA. Theobtained recombinant DNA is used to transform the Bacillus bacterium.

A gene derived from a Bacillus bacterium and a gene derived from otherorganisms such as Escherichia bacterium may be employed so long as thegene functions in Bacillus bacterium.

The targeted gene may be obtained, for example, by PCR using achromosomal DNA of a Bacillus bacterium as a template (PCR: polymerasechain reaction; White, T. J. et al., Trends Genet., 1989, 5, 185-189).Chromosomal DNA may be prepared from a bacterium serving as a DNA donorby the method of Saito and Miura (see H. Saito and K. Miura, Biochem.Biophys. Acta, 1963, 72, 619-629, Manual of Bioengineering Experiments,ed. by the Japan Bioengineering Society, pp. 97-98, Baifukan, 1992). Theprimers used in PCR can be designed based on the known sequence of thegene of Bacillus bacteria or based on the sequence conserved between thegenes from different organisms.

Examples of vectors capable of autonomous replicating used forintroducing target genes into a Bacillus bacterium include pUB110,pC194, and pE194. Furthermore, examples of vectors for introducingtarget genes into a chromosomal DNA include vectors for E. coli such aspHSG398 (Takara-Bio Co. (K.K.)) and pBluescript SK (Stratagene).

To prepare a recombinant DNA by ligating a target gene and marker into avector functioning in Bacillus bacterium, the vector is digested withsuitable restriction enzymes corresponding to the ends of the targetgene. For ligation, a ligase such as T4 DNA ligase can be employed.

The transformation methods as described above may be employed tointroduce the recombinant DNA prepared as described above into aBacillus bacterium. For example, competent cells can be prepared fromcells at the growing stage to introduce the DNA (Dubnau, D., andDavidoff-Abelson, R., J. Mol. Biol., 1971, 56, 209-221). Another methodwhere a recombinant DNA is incorporated into DNA-recipient cells in theform of protoplast or spheroplast that readily incorporates therecombinant DNA (Chang, S. and Cohen, S. N., Molec. Gen. Genet., 1979,168, 111-115) may also be used.

The copy number of the target gene can also be increased by integratingthe gene in multiple copies into the chromosomal DNA of a Bacillusbacterium. Multiple copies of a target gene can be integrated into thechromosomal DNA of a Bacillus bacterium by recombination using asequence present on the chromosomal DNA in multiple copies as a target.Examples of the sequences that are present in multiple copies onchromosomal DNA include transposons, repeat sequences, and invertedrepeats which are present on the ends of transposable elements.

In addition to the above-described gene amplification, the activity ofthe target enzyme can also be enhanced by replacing the expressionregulatory sequence of the promoter of the target gene on thechromosomal DNA or plasmid with a stronger one. The strength of apromoter is defined as the frequency of initiation acts of RNAsynthesis. Goldstein (Prokaryotic promoters in biotechnology.Biotechnol. Annu. Rev., 1995, 1, 105-128) discloses methods ofevaluating the strength of promoters and provides examples of strongpromoters. As disclosed in WO 00/18935, a promoter can be made strongerby replacing several nucleotides in the promoter region of the targetgene. Furthermore, substitution of nucleotides in the spacer regionbetween a ribosome binding site (RBS) and a start codon, particularlysubstitution of several nucleotides in the sequence immediately upstreamof a start codon, is known to strongly affect the translation efficiencyof mRNA. The modification of the expression regulatory sequence may becombined with increasing the copy number of the target gene.

Examples of promoters that function in Bacillus bacteria include the vegpromoter, spac promoter, and xyl promoter.

So long as the enzymatic activity of the oxidative pentosephosphatepathway is maintained, the gene encoding the enzyme of the oxidativepentosephosphate pathway may encode a protein having an amino acidsequence of SEQ ID NO: 48 or 50, wherein one or several amino acid aresubstituted, deleted, inserted, or added at one or multiple positions.Herein, although the term “several” depends on the type and position ofthe amino acid residues within the three dimensional structure of theprotein; it means 1 to 30, preferably 1 to 20, and more preferably, 1 to10.

The above-described mutation in the amino acid sequence of SEQ ID NO: 48or 50 protein is preferably a conservative mutation which does notimpair the enzymatic activity. A substitution means a mutation wherebyat least one residue in the amino acid sequence is removed and one ormore residues are inserted at that location. The conservativesubstitutions include: substitution of Ser or Thr for Ala, substitutionof Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Aspfor Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Seror Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln,substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Profor Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His,substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met,Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys,substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr,Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser,substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp,substitution of His, Phe, or Trp for Tyr and substitution of Met, Ile,or Leu for Val.

The above-described DNA encoding a protein substantially identical tothe wild-type enzyme of the oxidative pentosephosphate pathway can beobtained by a site-specific mutation method, for example, in which thenucleotide sequence encoding the enzyme is modified so as to substitute,delete, insert, add, or invert an amino acid residue at a specific site.The above-described modified DNA may also be obtained by a conventionalmutation treatment. Examples of mutation treatments include in vitrotreatment of a wild-type DNA with hydroxylamine and subjecting amicroorganism carrying a wild-type DNA, such as an Escherichia bacteriumtransformed with the DNA, to UV irradiation or treatment with amutagenic agent commonly employed in mutation treatments, such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid.

The DNA having the above-described mutation is induced in a suitablecell and the activity of the expression product is examined to obtain aDNA encoding a protein substantially identical to a wild-type enzyme ofthe oxidative pentosephosphate pathway. Furthermore, a DNA encoding sucha homolog can be obtained by hybridizing under stringent conditions witha probe having a part or all of the nucleotide sequence of SEQ ID NO: 47or 49, and wherein the DNA encodes a protein having an activity of anenzyme involved in the oxidative pentosephosphate pathway, and is from acell having the DNA encoding such a homolog enzyme. Herein, the term“stringent conditions” means conditions under which a specific hybridcan be formed and non-specific hybrids cannot be formed. Examplesthereof include conditions under which DNA fragments having a highhomology, for example, not less than 80%, preferably not less than 90%,and more preferably not less than 95% hybridize with each other, and DNAfragments having a lower homology do not hybridize. A specific examplethereof includes a condition of washing in Southern hybridization suchas a condition comprising washing at 1×SSC and 0.1% SDS, preferably0.1×SSC and 0.1% SDS at 60° C.

A portion of the nucleotide sequence of SEQ ID NO: 47 or 49 may be usedas a probe. Such a probe can be prepared by PCR using oligonucleotidesbased on the nucleotide sequence of SEQ ID NO: 47 or 49 as primers and aDNA fragment containing the nucleotide sequence of SEQ ID NO: 47 or 49as a template. When a DNA fragment of about 300 bp in length is used asa probe, the hybridization conditions may be washing at 50° C., 2×SSC,0.1% SDS.

A DNA encoding a protein substantially identical to a wild-type enzymeof the oxidative pentosephosphate pathway may encode a protein having ahomology of not less than 80%, preferably not less than 90%, and morepreferably not less than 95% to the amino acid sequence of SEQ ID NO: 48or 50, and which has an activity of an enzyme of the oxidativepentosephosphate pathway.

<2>Method for Producing a Purine-Derived Substance

The Bacillus bacterium of the present invention efficiently produces apurine-derived substance. Accordingly, purine-derived substances such aspurine nucleosides and purine nucleotides can be produced in a medium orin the bacterial cells by culturing the Bacillus bacterium of thepresent invention in a suitable medium.

The medium used for culturing the Bacillus bacterium of the presentinvention may be a common nutrient medium containing a carbon source,nitrogen source, inorganic salt source, and, if necessary, trace amountof organic nutrients such as amino acids and vitamins. The culture maybe performed according to conventional methods. Either a synthetic ornatural medium may be used. The carbon source and nitrogen source addedto the medium are not particularly limited so long as they can beassimilated by the Bacillus bacterium to be cultured.

The carbon source may be a sugar such as glucose, fructose, sucrose,maltose, mannose, galactose, arabinose, xylose, trehalose, ribose,starch hydrolysis products, and molasses; an alcohol such as glycerol ormannitol; an organic acid such as gluconic acid, acetic acid, citricacid, maleic acid, fumaric acid, and succinic acid. These carbon sourcesmay be used singly or in combination.

A nitrogen source may be ammonia, ammonium sulfate, ammonium carbonate,ammonium chloride, ammonium phosphate, ammonium acetate, other ammoniumsalts, nitrates, soybean hydrolysis products, and other forms of organicnitrogen may also be used.

Trace amounts of organic nutrients, such as amino acids, vitamins, fattyacids, nucleic acid, peptones containing these compounds, casaminoacids, yeast extract, and soy protein decomposition products, may beused. When a mutant strain auxotrophic for an amino acid, nucleoside, orthe like is cultured, it is necessary to supplement with the nutrientrequired by the strain.

Examples of inorganic salts include phosphate salts, magnesium salts,calcium salts, iron salts, manganese salts, and the like.

The culture conditions depend on the type of a Bacillus bacterium to becultured. In the case of Bacillus subtilis, culture may be conducted ata fermentation temperature of 20 to 50° C. with a regulated pH of 4 to 9and with aeration. When the pH decreases during the culture, the mediummay be neutralized with an alkali such as ammonium gas. Culture may beconducted for 40 hours to 3 days to produce a purine-derived substancein the culture medium.

The purine-derived substance, such as inosine, which is produced in theculture medium can be collected by a conventional method. For example,purine nucleosides including inosine and guanosine can be collected byprecipitation, ion-exchange chromatography, or the like.

Furthermore, the inosine or guanosine produced by the method of thepresent invention may be subjected to a reaction with purine nucleosidephosphorylase and phosphoribosyl transferase to obtain 5′-inosinic acidand 5′guanylic acid, respectively.

Furthermore, purine nucleotides (nucleoside-5′-phosphoric esters) can beproduced by subjecting the purine nucleoside produced by the method ofthe present invention to a reaction with a bacterium which has theability to produce nucleoside-5′-phosphoric ester, or with an acidphosphatase in the presence of a phosphate donor selected from the groupconsisting of polyphosphoric acid, phenyl phosphate, and carbamylphosphate. The bacterium which has the ability to producenucleoside-5′-phosphoric esters is not particularly limited so long asit has the ability to produce nucleoside-5′-phosphoric esters, andexamples thereof include a bacterium described in WO96/37603,Escherichia blattae JCM 1650, Serratia ficaria ATCC 33105, Klebsiellaplanticola IFO 14939 (ATCC 33531), Klebsiella pneumoniae IFO 3318 (ATCC8724), Klebsiella terrigena IFO 14941 (ATCC 33257), Morganella morganiiIFO 3168, Enterobacter aerogenes IFO 12010, Enterobacter aerogenes IFO13534 (ATCC 13048), Chromobacterium fluviatile IAM 13652,Chromobacterium violaceum IFO 12614, Cedecea lapagei JCM 1684, Cedeceadavisiae JCM 1685, and Cedecea neteri JCM 5909, disclosed inJP07-231793A.

An example of acid phosphatase (EC 3.1.3.2) which can be used forproducing purine nucleotides includes the one disclosed inJP2002-000289A, U.S. Pat. No. 6,010,851, U.S. Pat. No. 6,015,697,WO01/18184, and preferably includes a mutant acid phosphatase having anenhanced affinity for nucleosides (U.S. Pat. No. 6,015,697), a mutantacid phosphatase with no nucleotidase activity (WO96/37603), and amutant acid phosphatase with no phosphoric ester hydrolytic activity(U.S. Pat. No. 6,010,851).

EXAMPLES

The present invention is described in more detail by reference to thefollowing non-limiting examples.

Example 1

<Construction and Culture Evaluation of Purine NucleosidePhosphorylase-Disrupted Strain>

(1) Construction of a Purine Nucleoside Phosphorylase (pupG)-DisruptedStrain

A disrupted-type pupG gene was introduced into a recombinant KMBS16strain (2004-0166575A) which is derived from Bacillus subtilis (B.subtilis 168 Marburg strain; ATCC6051) and in which the purine operonrepressor gene (purR), succinyl-AMP synthase gene (purA), and purinenucleoside phosphorylase gene (deoD) are disrupted, as follows.

(i) Cloning of the 5′-End Region of the pupG Gene

Based on the information from GenBank (Accession No. NC_(—)000964), theprimers having the following nucleotide sequence were designed for PCR:

ATTGCACGGCCGTTCGTCGG (SEQ ID NO: 1)

cgcagatctCCGGATTTTCGATTTCGTCC (SEQ ID NO: 2; The nucleotide sequenceindicated by lower case letters is a tag containing a BglII site.)

Using a chromosomal DNA from B. subtilis 168 Marburg strain as atemplate and the above-described primers, PCR was conducted (94° C., 30seconds; 55° C., 1 minute; 72° C., 2 minutes; 30 cycles; Gene Amp PCRSystem Model 9600 (Perkins Elmer)), to amplify a fragment containing aregion upstream of a translation initiation codon of the pupG gene(about 610 bp) and its downstream region (about 120 bp).

The amplified fragment was purified by phenol-chloroform extraction andethanol precipitation. Perfectly Blunt Cloning Kit (NOVAGEN) wasemployed for cloning the fragment into the pT7Blue plasmid, which wasincluded in the kit. Both ends of the multicloning site of this plasmidare EcoRI and HindIII. A plasmid in which the pupG gene is inserted in adirection so that the upstream region of the pupG gene is on the EcoRIside was selected and named pKM48.

(ii) Cloning of the 3′-End Region of the pupG Gene

Based on the information from GenBank (Accession No. NC_(—)000964),primers having the following nucleotide sequence were designed for PCR:

CAAAGATCTGTCCAGCCTGG (SEQ ID NO: 3

cgcctgcagTGCCTTTATCTAAAGCTTCC (SEQ ID NO: 4; The nucleotide sequenceindicated by lower case letters is a tag containing a PstI site.)

Using a chromosomal DNA from B. subtilis 168 Marburg strain as atemplate and the above-described primers, PCR was conducted (94° C., 30seconds; 55° C., 1 minute; 72° C., 2 minutes; 30 cycles; Gene Amp PCRSystem Model 9600 (Perkins Elmer)), to amplify a fragment containing aregion upstream of the translation stop codon (about 340 bp) and itsdownstream region (about 400 bp).

In the same manner as the cloning of the 5′-end region of the pupG gene,the amplified fragment was cloned into pT7Blue plasmid, and thereby aplasmid in which the pupG gene is inserted in a direction so that thedownstream region of the pupG gene is on the EcoRI side was selected andnamed pKM49.

(iii) Cloning of ΔpupG Fragment

After digesting the pKM49 with SpeI, it was blunted with Klenow fragmentand further digested with PstI, to excise a DNA fragment of about 740bp. This fragment was ligated using T4 DNA ligase to a plasmid fragmentwhich was obtained by treating the pKM48 with BglII, blunting withKlenow fragment and digesting with PstI, and thereby the plasmid pKM75was obtained. This plasmid has about 1,470 bp insert comprising thedisrupted-type pupG fragment in which about 360 bp of the pupGstructural gene had been deleted.

(iv) Construction of a Plasmid to Disrupt the pupG Gene

An insert was excised from pKM75 by treating the plasmid with SacI andPstI. The obtained fragment was ligated using T4 DNA ligase to arecombination vector pJPM1 (Mueller et al., J. Bacteriol., 1992, 174,4361-4373) that had been treated with the same enzymes, and thereby aplasmid pKM76 was obtained.

Competent cells of the KMBS16 strain prepared by the method of Dubnauand Davidoff-Abelson (J. Mol. Biol., 1971, 56, 209-221) were transformedwith the plasmid pKM76, and the colonies (single-crossover recombinants)that were capable of growing on LB agar plate containing 2.5 μg/mL ofchloramphenicol were selected.

The obtained single-crossover recombinants were inoculated into 10 mL ofLB medium and successively subcultured for several days at 37° C.Colonies exhibiting chloramphenicol sensitivity were selected on a plateof LB medium including chloramphenicol. Chromosomal DNA was preparedfrom the obtained chloramphenicol-sensitive colonies. PCR was conductedin the same manner as described above using primers of SEQ ID NOS: 5 and6. Bacterial strains (purR::spc purA::erm deoD::kanΔpupG) in which thepupG gene on the chromosome had been replaced with the disrupted-typepupG gene (ΔpupG) by double-crossover recombination were identified. Theobtained strain was named KMBS93.

GGTCTGAGCTTTGCGAACC (SEQ ID NO: 5)

CGCCTGCAGTGCCTTTATCTAAAGCTTCC (SEQ ID NO: 6)

(2) The disrupted-type pupG gene was introduced into the recombinantKMBS13 strain (US2004-0166575A) which is derived from Bacillus subtilis(B. subtilis strain 168 Marburg; ATCC6051) and in which the purineoperon repressor gene (purR) and succinyl-AMP synthase gene (purA) aredisrupted, as follows.

Competent cells of the KMBS13 strain prepared as described above weretransformed with plasmid pKM76, and the colonies (single-crossoverrecombinants) that were capable of growing on an LB agar platecontaining 2.5 μg/mL of chloramphenicol were selected.

The obtained single-crossover recombinants were inoculated into 10 mL ofLB medium and successively subcultured for several days at 37° C.Colonies exhibiting chloramphenicol sensitivity were selected on a plateof LB medium including chloramphenicol. Chromosomal DNA was preparedfrom the obtained chloramphenicol-sensitive colonies. PCR was conductedin the same manner as described above using primers of SEQ ID NOS: 5 and6. Bacterial strains (purR::spc purA::erm ΔpupG) in which the pupG geneon the chromosome had been replaced with the disrupted-type of pupG gene(ΔpupG) by double-crossover recombination were identified. The obtaineddouble-recombinant strain was named KMBS113.

(3) Purine Nucleoside Production by the pupG Gene-Disrupted Strains

The pupG gene-disrupted strains (KMBS93 and KMBS113) and control strain(KMBS16) were uniformly spread over PS plate medium (30 g/L of solublestarch, 5 g/L of yeast extract, 5 g/L of polypeptone, 20 g/L of agar,adjusted to pH 7.0 with KOH) and cultured overnight at 34° C. One-eighthof the bacterial cells on the plate were inoculated into 20 mL offermentation medium contained in a 500 mL capacity Sakaguchi flask.Subsequently, calcium carbonate was added at 50 g/L and culture wasperformed at 34° C. with shaking. Sampling was conducted at the timepoint of 100 hours from the start of culturing, and the amounts ofinosine and hypoxanthine accumulated in the culture medium were measuredby conventional methods. A large amount of hypoxanthine was detected inthe culture medium of the control KMBS16 strain. However, thehypoxanthine accumulation by the pupG gene-disrupted strains KMBS93 andKMBS113 was extremely low, and no clear peak was detected by HPLC (Table1). On the other hand, the inosine accumulation by the strains KMBS93and KMBS113 was greater than that of the control strain KMBS16.

Composition of the fermentation medium: Glucose 80 g/L KH₂PO₄ 1 g/LNH₄Cl 32 g/L Mameno (T-N)* 1.35 g/L DL-methionine 0.3 g/L L-tryptophan0.02 g/L Adenine 0.1 g/L MgSO₄ 0.4 g/L FeSO₄ 0.01 g/L MnSO₄ 0.01 g/LGD113 0.01 mL/L (adjusted to pH 7.0 with KOH) Calcium carbonate 50 g/L*Protein hydrolysis product

TABLE 1 B. subtilis strain OD562 Hypoxanthine g/L Inosine g/L KMBS168.84 2.27 1.65 KMBS93 7.02 ND*¹ 4.49 KMBS113 7.02 ND*¹ 5.43*¹No clear peak could be detected under the HPLC conditions employed.

Example 2

(1) Introduction of a Mutant guaB Gene

A guaB (A1) mutation which results in replacement of the Alanine atposition 226 in SEQ ID NO: 62 with Valine in the IMP dehydrogenase gene(guaB) was introduced into a recombinant KMBS113 strain that was derivedfrom Bacillus subtilis (B. subtilis strain 168 Marburg; ATCC6051) and inwhich the purine operon repressor gene (purR), succinyl-AMP synthasegene (purA), and purine nucleosidase phosphorylase gene (pupG) had beendisrupted, as follows. The introduction of the guaB(A1) mutation causesa reduction in the enzyme activity of IMP dehydrogenase.

(i) Preparation of a Bacterial Strain in Which a Kanamycin-ResistanceGene was Inserted into the Middle of the Wild-Type guaB Gene Derivedfrom B. subtilis Strain 168 Marburg Strain

In amplification of the upstream region of the guaB gene, PCR primershaving the nucleotide sequences shown below were designed based onGenBank information (Accession No. NC_(—)000964 and V01547):

cgcggatccGGCTTAACGTTCGACGATGTGCTGC (SEQ ID NO: 7; The nucleotidesequence indicated by lower case letters is a tag containing the BamHIsite);

gctttgcccattctatagatatattGAGTCATTGTATCGCCAGTTACACC (SEQ ID NO: 8; Thenucleotide sequence indicated by lower case letters is the sequenceupstream of the promoter of the kanamycin-resistance gene (kan) whichwas cloned into pDG783 (BGSC)).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primer and a template of a chromosomalDNA of B. subtilis 168 Marburg strain to amplify a fragment (about 710bp) of the 5′-end of the guaB gene.

In amplification of the 3′-end of guaB gene, PCR primers having thenucleotide sequences shown below were designed based on GenBankinformation (Accession No. NC_(—)000964 and V01547):

cctagatttagatgtctaaaaagctGTGATTGTTATCGATACAGCTCACG (SEQ ID NO: 9; thenucleotide sequence indicated by lower case letters is the sequencedownstream of the structural gene of the kanamycin-resistance gene (kan)which was cloned into pDG783 (BGSC);

cgcgaattcGTAATCTGTACGTCATGCGGATGGC (SEQ ID NO: 10: the nucleotidesequence represented in lower case letters is a tag containing the EcoRIsite).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe above-described primers and a template of chromosomal DNA of B.subtilis 168 Marburg strain to obtain an amplified fragment (about 730bp) of 3′-end of the guaB gene.

In amplification of the 3′-end of kan gene by PCR, PCR primers havingthe following nucleotide sequence were prepared based on informationfrom GenBank (Accession Nos. V01277 and NC_(—)000964):

ggtgtaactggcgatacaatgactcAATATATCTATAGAATGGGCAAAGC (SEQ ID NO: 11; thenucleotide sequence indicated by lower case letters is complementary tothe 3′-end region of the sequence of the guaB upstream region in SEQ IDNO: 8;);

cgtgagctgtatcgataacaatcacAGCTTTTTAGACATCTAAATCTAGG (SEQ ID NO: 12; thenucleotide sequence indicated by lower-case letters is complementary tothe 3′-end region of the guaB downstream sequence in SEQ ID NO: 9).

PCR (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1 minute; 30 cycles;Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using theabove-described primers and a template of the plasmid DNA containing akanamycin-resistance gene (kan), such as pDG783, to amplify a fragmentof about 1,150 bp which contains the kan gene.

In amplification of the guaB region inserted with the kan gene byrecombinant PCR, the three DNA fragments amplified as described abovewere purified using MicroSpin Column S-400 (Amersham Pharmacia Biotech),a suitable quantity of the mixture was used as a template, and theprimers of SEQ ID NOS: 7 and 10 were used, and PCR (94° C., 30 seconds;55° C., 1 minute; 72° C., 2 minutes; 30 cycles; Gene Amp PCR SystemModel 9600 (made by Perkins Elmer)) was conducted, and thereby anamplified fragment of the guaB gene into which a kan gene had beeninserted was obtained.

The target fragment of the guaB gene into which a kan gene had beenintroduced (guaB::kan) was extracted from the gel following agarose gelelectrophoresis. Competent cells of B. subtilis strain prepared asdescribed above were transformed with the DNA fragment and colonies thatwere capable of growing on an LB agar plate containing 2.5 μg/mL ofkanamycin and 20 μg/mL of guanine were selected. Chromosomal DNA wasprepared from these colonies and PCR was conducted using primers of SEQID NOS: 7 and 10 to identify a bacterial strain in which the native guaBregion on the chromosome had been replaced with a guaB gene in which theinternal sequence had been inserted with a kanamycin-resistance gene(guaB::kan) by double-crossover recombination. The recombinant strainthus obtained was guanine-auxotroph; the strain was named KMBS193.

(ii) Preparation of a Bacterial Strain Which is Derived from B. subtilis168 Marburg and in Which a guaB Mutation (A1) is Introduced

In amplification of the 5′-end of guaB gene, PCR primers having thefollowing nucleotide sequences were designed based on the informationfrom GenBank (Accession No. NC_(—)000964):

CATAAAATGTACGCATAGGCCATTC (SEQ ID NO: 13);

TTGTATCGCCAGTTACACCAACTaCCGCGCCAACGATCAGGCGGCC (SEQ ID NO: 14: thenucleotide indicated by a lower-case letter is a mutation point).

PCR (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1 minute; 30 cycles;Gene Amp PCR System Model 9600 (Perkins Elmer)) was conducted using theabove-described primers and a template of the chromosomal DNA of B.subtilis 168 Marburg strain to amplify a fragment (about 1,210 bp)containing the 5′-end region and the upstream region of the guaB gene.

Then, the 3′-end of guaB gene was amplified. Based on the informationfrom GenBank (Accession No. NC_(—)000964), PCR primers having thefollowing nucleotide sequences were prepared:

GGCCGCCTGATCGTTGGCGCGGtAGTTGGTGTAACTGGCGATACAA (SEQ ID NO: 15: thenucleotide indicated by a lower-case letter is a mutation point; thisprimer is complementary to SEQ ID NO: 14);

CCTTGATCAATTGCTTCCAATAACAG (SEQ ID NO: 16)

PCR (94° C., 30 second; 55° C., 1 minute; 72° C., 1 minute; 30 cycles;Gene Amp PCR System Model 9600 (made by Perkins Elmer)) was conductedusing the above-described primers and a template of the chromosomal DNAof B. subtilis 168 Marburg strain to amplify a fragment (about 1,220 bp)containing 3′-end and downstream region of the guaB gene.

In amplification of the guaB region into which a guaB (A1) mutation hadbeen introduced by recombinant PCR, the two DNA fragments obtained asdescribed above were subjected to agarose gel electrophoresis and thetarget DNA fragments were purified. A mixture of suitable quantities ofthe two DNA fragments was employed as a template, SEQ ID NOS: 13 and 16were employed as primers, and PCR (94° C., 30 second; 55° C., 1 minute;72° C., 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (made byPerkins Elmer)) was conducted to amplify a fragment of the guaB geneinto which a guaB (A1) mutation had been introduced.

The target fragment of the guaB gene (guaB (A1)) into which a guaB (A1)mutation had been introduced was extracted from the gel followingagarose gel electrophoresis. Competent cells of KMBS193 prepared asdescribed above were transformed with the DNA fragment, and coloniesthat were capable of growing on minimal medium agar plate were selected.Chromosomal DNA was prepared from these colonies and PCR was conductedwith primers of SEQ ID NOS: 13 and 16 to identify bacterial strains inwhich the guaB::kan region on the chromosome had been replaced with aguaB region containing a guaB (A1) mutation by double-crossoverrecombination. The recombinant thus obtained was not guanine-auxotroph;and the strain was named YMBS9.

(iii) Preparation of a Bacterial Strain Derived from Inosine-ProducingBacterium KMBS113 and into Which a guaB Mutation (A1) was Introduced

Chromosomal DNA of KMBS193 (guaB::kan) was prepared and used totransform competent cells of strain KMBS113 prepared as described above,and colonies that were capable of growing on an LB agar plate containing2.5 μg/mL of kanamycin and 20 μg/mL of guanine were selected. Theobtained strain was auxotrophic for guanine, and named YMBS6 (purR::spcpurA::erm ΔpupG guaB::kan trpC2).

Next, a chromosomal DNA of YMBS9 (guaB(A1)) was prepared and used totransform competent cells of strain YMBS6 prepared as described above,and colonies that were capable of growing on a minimal medium agar platecontaining 20 μg/mL of adenine were selected. The obtained strain wasleaky auxotrophic for guanine, and the strain was named YMBS2 (purR::spcpurA::erm guaB(A1) ΔpupG trpC2).

(2) Production of Purine-Derived Nucleic Acid by an Inosine-ProducingStrain into Which the guaB Mutation was Incorporated

An inosine-producing strain into which guaB (A1) mutation had beenincorporated (YMBS2 strain) and a control KMBS113 strain were uniformlyspread over PS plate medium (soluble starch 30 g/L, yeast extract 5 g/L,polypeptone 5 g/L, agar 20 g/L, adjusted to pH 7.0 with KOH) andcultured overnight at 34° C. One-eighth of the bacterial cells wereinoculated into 20 mL of fermentation medium in a 500 mL capacitySakaguchi flask. Subsequently, calcium carbonate was added at 50 g/L andeach of the bacteria was cultured at 34° C. with shaking. Sampling wasconducted at the time point of 96 hours after the start of culturing,and the amounts of inosine and hypoxanthine in the culture medium weremeasured by conventional methods.

Composition of the fermentation medium: Glucose 60 g/L KH₂PO₄ 1 g/LNH₄Cl 32 g/L Mameno (T-N)* 1.35 g/L DL-methionine 0.3 g/L L-tryptophan0.02 g/L Adenine 0.1 g/L MgSO₄ 0.4 g/L FeSO₄ 0.01 g/L MnSO₄ 0.01 g/LGD113 0.01 mL/L (adjusted to pH 7.0 with KOH) Calcium carbonate 50 g/L*Protein hydrolysis product

TABLE 2 B. subtilis strain OD610 Inosine (g/L) Xanthosine (g/L) KMBS11317.4 3.1 0.06 YMBS2 6.43 3.7 0.05

Example 3

<Preparation of a Purine Operon-Amplified Strain>

(1) Preparation of Ppur-Disrupted Strain

A strain derived from Bacillus subtilis (B. subtilis 168 Marburg strain;ATCC6051) and in which purine operon promoter (Ppur) was disrupted wasprepared as follows.

(i) Amplification of the Upstream Region of the Ppur by PCR

Based on the information from GenBank (Accession No. NC_(—)000964 andV01277), PCR primers having the following nucleotide sequence wereprepared:

cgcggatccTTATTTAGCGGCCGGCATCAGTACG (SEQ ID NO: 17; the nucleotidesequence indicated by lower-case letters is a tag containing a BamHIsite);

cgtttgttgaactaatgggtgctttATGGATAATGTCAACGATATTATCG (SEQ ID NO: 18: thenucleotides indicated by lower-case letters is the sequence of upstreamof the promoter of the chloramphenicol-resistance gene (cat) that iscloned into pC194).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe above-described primers and a template of chromosomal DNA of the B.subtilis 168 Marburg strain to amplify a fragment containing the Ppur-35sequence and the upstream region (about 730 bp).

(ii) Amplification of the Downstream Region of Ppur by PCR

Based on the information from GenBank (Accession No. NC_(—)000964 andV01277), PCR primers having the following nucleotide sequences wereprepared:

acagctccagatccatatccttcttCCTCATATAATCTTGGGAATATGGC (SEQ ID NO: 19; thenucleotide sequence indicated by lower-case letters is the sequence ofdownstream region of the structural gene of thechloramphenicol-resistance gene (cat) that is cloned into the pC194plasmid);

cgcggatccTCTCTCATCCAGCTTACGGGCAAGG (SEQ ID NO: 20; the nucleotidesequence indicated by lower-case letters is a tag containing the BamHIsite).

PCR was conducted (94° C., 30 second; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe above-described primers and a template of a chromosomal DNA of B.subtilis 168 Marburg strain to amplify a fragment containing about 230bp region upstream of the purE gene translation start codon and itsdownstream region of about 440 bp.

(iii) Amplification of the Cat Gene by PCR

PCR primers having the following sequences were designed based on theinformation from GenBank (Accession No. V01277 and NC_(—)000964):

cgataatatcgttgacattatccatAAAGCACCCATTAGTTCAACAAACG (SEQ ID NO: 21; thenucleotide sequence indicated by lower-case letters is complementary tothe 3′-end region of the Ppur upstream region sequence in SEQ ID NO:18);

gccatattcccaagattatatgaggAAGAAGGATATGGATCTGGAGCTGT(SEQ ID NO: 22; thenucleotide sequence indicated by lower-case letters is complementary tothe 3′-end of the region upstream of translation start codon in the purEgene in SEQ ID NO. 19).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 2minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of the plasmidDNA comprising a chloramphenicol-resistance gene (cat), such as pC194,to amplify a fragment of about 980 bp containing the cat gene.

(iv) Amplification of the Ppur Region in Which a Cat Gene is Inserted byPCR

The DNA fragments amplified in the above (i) to (iii) were purified on aMicroSpin Column S-400 (Amersham Pharmacia Biotech), mixed in suitablequantities, and used as templates; primers of SEQ ID NOS: 17 and 20 wereemployed; and PCR was conducted (94° C., 30 seconds; 55° C., 1 minute;72° C., 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (made byPerkins Elmer)), and thereby a fragment of the Ppur region in which acat gene is inserted was obtained.

(v) Production of a Bacterial Strain in Which Ppur is Disrupted

The DNA fragment of the Ppur region in which cat gene is inserted(Ppur::cat) obtained in the above (iv) was subjected to agarose gelelectrophoresis and the fragment was extracted from the gel. DNAfragment purified in this manner was used to transform competent cellsof B. subtilis prepared as described above, and the colonies that werecapable of growing on LB agar plate containing 2.5 μg/mL ofchloramphenicol were selected. Chromosomal DNA was prepared from thesecolonies. Using the PCR fragment described in the above (iv), abacterial strain in which the chromosomal Ppur region had been replacedwith a Ppur region into which cat gene had been inserted (Ppur::cat) bydouble-crossover recombination was identified. The recombinant strainobtained in this manner was auxotrophic for adenine; and the strain wasnamed KMBS198.

(2) <Preparation of a Purine Operon Promoter Mutant>

A bacterial strain derived from Bacillus subtilis (B. subtilis 168Marburg strain; ATCC6051) and modified to change the −10 sequence ofPpur to the similar consensus sequence TATAAT (Ppur1), TATGAT (Ppur3),and TAAAAT (Ppur5), respectively was prepared as follows.

(i) Amplification of the Upstream Region Containing the “−10” Sequenceof the Ppur by PCR

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimer of SEQ ID NO: 42 and each of the primers (SEQ ID NO: 23-25)containing three kinds of the modified Ppur sequence were designed:

AATAGATATAAAGAGGTGAGTCTGC (SEQ ID NO: 42)

<for Ppur1 Modification>

TTTTGATTTCATGTTTattataACAACGGACATGGATA (SEQ ID NO: 23; the nucleotidesequence indicated by lower-case letters is the Ppur1 sequence)

<for Ppur3 Modification>

TTTTGATTTCATGTTTatcataACAACGGACATGGATA (SEQ ID NO: 24; the nucleotidesequence indicated by lower-case letters is the Ppur3 sequence)

<for Ppur5 Modification>

TTTTGATTTCATGTTTattttaACAACGGACATGGATA (SEQ ID NO: 25; the nucleotidesequence indicated by lower-case letters is the Ppur5 sequence).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of achromosomal DNA of B. Subtilis 168 Marburg strain, to amplify threekinds of fragments of about 1,060 bp containing Ppur and its upstreamsequence.

(ii) Amplification of the Downstream Region Containing the Modified“−10” Sequence of Ppur by PCR

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimer of SEQ ID NO: 26 and each of the primers containing the threekinds of modified Ppur sequences were prepared:

GGGTAATAAGCAGCAGCTCACTTCC (SEQ ID NO: 26)

<for Ppur1 Modification>

TATCCATGTCCGTTGTtataatAAACATGAAATCAAAA (SEQ ID NO: 27; the nucleotidesequence indicated by lower-case letters is the Ppur 1 sequence, thisprimer is complementary to SEQ ID NO: 23)

<for Pur3 Modification>

TATCCATGTCCGTTGTtatgatAAACATGAAATCAAAA (SEQ ID NO: 28; the nucleotidesequence indicated by lower-case letters is the Ppur 3 sequence, thisprimer is complementary to SEQ ID NO: 24)

<for Pur5 Modification>

TATCCATGTCCGTTGTtaaaatAAACATGAAATCAAAA (SEQ ID NO: 29: the nucleotidesequence indicated by lower-case letters is the Ppur 5 sequence, thisprimer is complementary to SEQ ID NO: 25)

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of achromosomal DNA of B. Subtilis 168 Marburg strain, to amplify threekinds of fragments of about 1,070 bp containing the Ppur “−10” sequenceand its downstream sequence.

(iii) Amplification of the Ppur Region Containing the Modified “−10”Sequence by PCR

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 2minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using primers of SEQ ID NOS: 42 and 26 and a template of each ofthe DNA fragments amplified in the above (i) and (ii) and purified on aMicroSpin Column S-400 (Amersham Pharmacia Biotech), to amplifyfragments of the Ppur region in which the “−10” sequence had beenreplaced with Ppur1, Ppur3, and Ppur5, respectively.

(iv) Preparation of a Bacterial Strain in Which the “−10” Sequence isReplaced by Each of Ppur1, Ppur3, and Ppur5

DNA fragments of the Ppur region containing the modified “−10” sequence(Ppur1, Ppur3, or Ppur5) obtained in the above (iii) were subjected toagarose gel electrophoresis and the target fragments were extracted fromthe gel. Competent cells of strain KMBS 198 prepared as described abovewere transformed with the obtained DNA fragments, and colonies that werecapable of growing on minimal medium agar plate were selected.Chromosomal DNA was prepared from these colonies and the strains inwhich the Ppur::cat region on the chromosome had been replaced by Ppur1,Ppur3, and Ppur5 by double-crossover recombination were identified bythe PCR as described in the above (iii). The recombinants thus obtainedwere not auxotrophic for adenine. The obtained strains were namedKMBS210, KMBS211, and KMBS222, respectively.

(3) Preparation of a Strain in Which an Attenuator Sequence of thePurine Operon is Deleted

A bacterial strain in which the attenuator sequence located downstreamof Ppur was deleted was prepared from Bacillus subtilis (B. subtilis 168Marburg strain; ATCC6051) as follows. FIG. 1 shows the sequence that wasdeleted.

(i) Amplification of the Partially Deleted Attenuator Sequence and itsUpstream Region by PCR

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimer of SEQ ID NO: 17 and the primer of SEQ ID NO: 30 having thefollowing nucleotide sequence were prepared:

GCTTTTGTTTTCAGAAAATAAAAAATAcgATATATCCATGTCAGTTTTATCG (SEQ ID NO: 30, thenucleotide sequence indicated by lower-case letters is a junctioncreated by deleting a portion (75 bp) of the attenuator sequence).

PCR was conducted (94° C., 30 seconds; 53° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of achromosomal DNA of B. subtilis 168 Marburg strain to amplify a fragmentcontaining a partially deleted attenuator sequence and its upstreamsequence (about 840 bp).

(ii) Amplification of the Partially Deleted Attenuator Sequence and itsDownstream Region by PCR

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimer of SEQ ID NO: 26 and the primer of SEQ ID NO: 31 having thefollowing nucleotide sequence were prepared:

CGATAAAACTGACATGGATATATcgTATTTTTTATTTTCTGAAAACAAAAGC (SEQ ID NO: 31; thenucleotide sequence indicated by lower-case letters is a junctioncreated by deleting a part (75 bp) of the attenuator sequence. Thisprimer is complementary to SEQ ID NO: 30).

PCR was conducted (94° C., 30 seconds; 53° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe above-described primers and a template of a chromosomal DNA of B.subtilis 168 Marburg strain to amplify a fragment containing a partiallydeleted attenuator sequence and its downstream sequence (about 850 bp).

(iii) Amplification of a Ppur Region Containing a Partially DeletedAttenuator Sequence by PCR

The DNA fragments amplified in the above (i) and (ii) were purified on aMicroSpin Column S-400 (Amersham Pharmacia Biotech), and a suitablequantity of the mixture was used as template, and primers of SEQ ID NOS:17 and 26 were used, and PCR (94° C., 30 seconds; 53° C., 1 minute; 72°C., 2 minutes; 30 cycles; Gene Amp PCR System Model 9600 (PerkinsElmer)) was conducted, to amplify a fragment of the Ppur regioncontaining a partially deleted attenuator sequence.

(iv) Construction of a Bacterial Strain with a Partially DeletedAttenuator Sequence

The DNA fragment of the Ppur region (Ppur-Δatt) containing the partiallydeleted attenuator sequence obtained in the above (iii) was subjected toagarose gel electrophoresis and the target fragment was extracted fromthe gel. Competent cells of the strain KMBS198 prepared as describedabove were transformed with the DNA the obtained fragment, and thecolonies that were capable of growing on a minimal medium agar platewere selected. Chromosomal DNA was prepared from these colonies and thestrain in which the Ppur::cat region on the chromosome had beensubstituted with Ppur-Δatt by double-crossover recombination wasidentified by the PCR as described in the above (iii). The obtainedstrain was auxotroph for adenine. The strain was named KMBS252.

(4) A bacterial strain derived from Bacillus subtilis (B. subtilisstrain 168 Marburg; ATCC6051) that was modified by changing the “−10”sequence of Ppur to the consensus sequence TATAAT (Ppur1) and partiallydeleting the attenuator sequence was prepared as follows.

(i) Amplification of the Upstream Region Containing the Modified “−10”Sequence (Ppur1) of Ppur by PCR

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe primers of SEQ ID NOS: 42 and 23 designed based on GenBank AccessionNo. NC_(—)000964 and a template of the chromosomal DNA of a bacterialstrain in which the attenuator sequence had been partially deleted suchas the KMBS252 strain prepared in the above (3), to amplify a fragmentof about 1060 comprising the upstream region of Ppur containing Ppur1.

(ii) Amplification of a Fragment Containing Ppur1, a Partially DeletedAttenuator Region, and its Downstream Region by PCR

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the primers of SEQ ID NOS: 26 and 27 designed based onGenBank Accession No. NC_(—)000964 and a template of the chromosomal DNAof a bacterial strain in which the attenuator sequence had beenpartially deleted such as the KMBS252 strain prepared in the above (3),to amplify a fragment of about 990 bp containing Ppur1, a partiallydeleted attenuator region, and the downstream region.

(iii) Amplification of a Fragment Containing the Ppur Region ContainingPpur1 and a Partially Deleted Attenuator Region by PCR

The DNA fragments amplified in the above (i) and (ii) were purified on aMicroSpin Column S-400 (Amersham Pharmacia Biotech), and a suitablequantity of the mixture was used as template, and primers of SEQ ID NOS:42 and 26 were used, and PCR (94° C., 30 seconds; 55° C., 2 minute; 72°C., 2 minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) was conducted, to amplify a fragment of the Ppur regioncontaining Ppur1 and a partially deleted attenuator sequence.

(iv) Preparation of a Bacterial Strain in which the “−10” Sequence wasChanged to Ppur1 and the Attenuator Sequence was Partially Deleted

The DNA fragment of the Ppur region (Ppur1-Δatt) containing Ppur1 and apartially deleted attenuator sequence obtained in the above (iii) wassubjected to agarose gel electrophoresis and the target fragment wasextracted from the gel. Competent cells of strain KMBS198 prepared asdescribed above were transformed with the obtained DNA fragment, and thecolonies that were capable of growing on minimal medium agar plate wereselected. Chromosomal DNA was prepared from these colonies and thestrains in which the Ppur::cat region on the chromosome had beensubstituted with Ppur1-Δatt by double-crossover recombination wereidentified by PCR as described in the above (iii). The obtainedrecombinant strain was not auxotrophic for adenine. The strain was namedKMBS261.

(5) Construction of a Plasmid for Measuring Transcription Activity ofPurine Operon

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimers having the following nucleotide sequences were prepared:

cgcaagcttTATTTTCTGAAAACAAAAGC (SEQ ID NO: 32; the nucleotide sequenceindicated by lower-case letters is a tag containing a HindIII site);

cgcggatccTTTCTCTTCTCTCATCCAGC (SEQ ID NO: 33: the nucleotide sequenceindicated by lower-case letters is a tag containing a BamHI Site).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 2minutes; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of achromosomal DNA of B. subtilis 168 Marburg strain to amplify a fragmentcontaining a part of the purE structural gene (445 bp) and its upstreamregion (40 bp) containing an SD sequence.

The amplified fragment was purified with a MinElute PCR Purification Kit(Qiagen) and digested with restriction enzymes at HindIII and BamHI. TheDNA fragment was inserted using T4 DNA ligase into the upstream regionof lacZ in pMutin4 (Vagner, V., et al., Microbiology, 1998, 144,3097-3104) that had been treated with the same enzymes, and thereby aplasmid pKM191 was obtained. pKM191 contains a part of the purEstructural gene (445 bp) and its upstream region (40 bp) containing anSD sequence.

(6) Preparation of a Bacterial Strain for Measuring the TranscriptionActivity of Purine Operon

A bacterial strain for measurement of transcription activity of purineoperon was constructed as follows.

(i) Preparation of a Bacterial Strain in Which Ppur is Disrupted

Chromosomal DNA was prepared from KMBS198 (Ppur::cat), and used totransform competent cells of KMBS4 strain (JP2004-242610A) which has adisrupted-type of purR (purR::spc) prepared as described above, and thecolonies that were capable of growing on LB agar plate containing 2.5μg/mL of chloramphenicol were selected. The obtained recombinant strainwas purR-deficient and auxotrophic for adenine. The strain was namedKMBS278.

(ii) Construction of a Bacterial Strain having a purR-Deletion and theModified Ppur Region

The chromosomal DNA was prepared from the strains KMBS210, KMBS211,KMBS222, KMBS252, and KMBS261, respectively. This DNA was used totransform competent cells of strain KMBS278 prepared as described above,and the colonies that were capable of growing on a minimal medium agarplate and exhibit spectinomycin-resistance were selected by using LBagar plate. Chromosomal DNA was prepared from these colonies and thestrains in which the Ppur::cat region on the chromosome had beensubstituted with a modified Ppur region by double-crossoverrecombination were identified by PCR as described in the above (1)(iv).The obtained recombinant strains were not auxotrophic for adenine. Thestrains were named KMBS283, KMBS284, KMBS285, KMBS286, and KMBS287,respectively.

(iii) Preparation of a Bacterial Strain in Which pKM191 is Introduced

Competent cells of each of B. subtilis 168 Marburg strain, KMBS4,KMBS283, KMBS284, KMBS285, KMBS286, and KMBS287 prepared as describedabove were transformed with plasmid pKM191 for measurement oftranscription activity of purine operon, and colonies that were capableof growing on LB agar plate containing 2.5 μg/mL of chloramphenicol wereselected. The obtained colonies were single crossover recombinants, inwhich native purE gene was recombined with purE gene of pKM191containing lacZ gene by homologous recombination, and turned blue on LBagar plate containing 80 μg/mL of X-Gal when lacZ gene is expressed byPpur. The strains were named KMBS292, KMBS295, KMBS296, KMBS297,KMBS298, KMBS299, and KMBS300, respectively.

(7) Measurement of Transcription Activity of Purine Operon

Ppur transcription activity was measured based on lacZ by using thebacterial strains prepared in the above (6) (iii). The KMBS292 strainhaving gene structure of 168 Marburg and the KMBS295 strain having genestructure of a purine operon repressor purR gene-disrupted were used ascontrol strains. The other strains had purR gene-disrupted background,and among them, the strains KMBS296, KMBS297, and KMBS298 have themodified “−10” sequences, and the KMBS299 strain has a partially deletedattenuator sequence, and the KMBS300 strain has both the modified “−10”sequence (Ppur1) and a partially deleted attenuator sequence.

These six strains were cultured in LB medium (20 mL) containing guanine(20 mg/L) at 37° C. until the late log growth phase (OD₆₀₀=1.1 to 1.4).A suitable quantity of the culture solution was sampled andβ-galactosidase activity therein was measured. The β-galactosidaseactivity was measured by the method of Fouet et al. (Fouet, A. andSonenshein, A. L., J. Bacteriol., 1990, 172, 835-844). The results areshown in FIG. 2. (WT shows the activity of KMBS296, ΔRpurR shows theactivity of KMBS295, ΔR+Ppur1 shows the activity of KMBS296, ΔR+Ppur3shows the activity of KMBS297, ΔR+Ppur5 shows the activity of KMBS298,ΔR+Ppur-Δatt shows the activity of KMBS299, andΔR+Ppur1-Δatt theactivity of KMBS300. The activity in the ΔpurR strain increased to4.5-fold as compared to the 168 Marburg strain. Furthermore,modification of the “−10” sequence to Ppur1 increased the activity about3-fold. Partial deletion of the attenuator sequence resulted in about15-fold increase in the activity, and in the strain having both of themutations, the activity increased to 26.5-fold.

Example 4

<Construction and Evaluation of Strains in Which the Modified PpurRegion is Introduced>

(1) The modified purine operon promoter and the partially deletedattenuator sequence were introduced into a recombinant YMBS2 strain thatis derived from Bacillus subtilis (B. subtilis 168 Marburg strain;ATCC6051) and in which the purine operon repressor gene (purR), succinylAMP synthase gene (purA), and purine nucleoside phosphorylase gene(pupG) had been disrupted and a guanine-auxotrophic leaky mutation(guaB(A1) mutation) had been introduced into the IMP dehydrogenase gene.

A chromosomal DNA was prepared from B. subtilis 168 Marburg strain, andused to transform the competent cells of KMBS113 strain (purR::spcpurA::erm ΔpupG trpC2) prepared as described above, and the coloniesthat were capable of growing on minimal medium agar plate (that is, notauxotrophic for adenine) and exhibited spectinomycin-resistance wereselected by using LB agar plate. Chromosomal DNA was prepared from thesecolonies, and among them, ΔpupG strain was identified by PCR as in theExample 1 (1)(iv). The obtained strain was named KMBS180 (purR::spcΔpupG trpC2).

Next, a chromosomal DNA of strain KMBS198 was prepared and used totransform competent cells of strain KMBS 180 prepared as describedabove, and the colonies that were capable of growing on an LB agar platecontaining 2.5 μg/mL of chloramphenicol were selected. Chromosomal DNAwas prepared from these colonies, and among them, the strain in whichthe Ppur region on the chromosome had been substituted with Ppur::cat bydouble-crossover recombination was identified by PCR in the same way asin Example 3(1)(iv). The strain was auxotrophic for adenine; the strainwas named KMBS216 (Ppur::cat purR::spc ΔpupG trpC2).

Chromosomal DNA of KMBS252 (Ppur-Δatt) was prepared and used totransform competent cells of strain KMBS216 prepared as described above,and the colonies that were capable of growing on a minimal medium agarplate were selected. Chromosomal DNA was prepared from these colonies,and among them, the bacterial strain in which the Ppur::cat on thechromosome had been substituted with the Ppur-Δatt region bydouble-crossover recombination was identified by PCR in the same way asin Example 3(1)(iv). The bacterial strain was not auxotrophic foradenine; the strain was named KMBS264 (Ppur-Δatt purR::spc ΔpupG trpC2).KMBS261, which is a bacterial strain in which Ppur1-Δatt is introduced,was obtained by the same procedure.

Chromosomal DNA of KMBS9 (purA::erm trpC2; US2004-0166575) was preparedand used to transform competent cells of strains KMBS264 and KMBS261prepared as described above, and the colonies that were capable ofgrowing on LB agar plate containing 0.5 μg/mL of erythromycin wereselected. These strains were not auxotrophic for adenine; and thestrains were named KMBS265 (Ppur-Δatt purR::spc purA::erm ΔpupG trpC2)and KMBS267 (Ppur1-Δatt purR::spc purA::erm ΔpupG trpC2), respectively.

Next, chromosomal DNA of KMBS193 (guaB::kan) was prepared and used totransform competent cells of strains KMBS265 and KMBS267 prepared asdescribed above, and the colonies that were capable of growing on LBagar plate containing 2.5 μg/mL of kanamycin and 20 μg/mL of guaninewere selected. These strains were auxotrophic for guanine; and thesestrains were named KMBS270 (Ppur-Δatt purR::spc purA::erm guaB::kanΔpupG trpC2) and KMBS271 (Ppur1-Δatt purR::spc purA::erm guaB::kan ΔpupGtrpC2), respectively.

Finally, chromosomal DNA of YMBS2 (guaB(A1)) was prepared and used totransform competent cells of strains KMBS270 and KMBS271 prepared asdescribed above, and the colonies that were capable of growing onminimal medium agar plate containing 20 μg/mL of adenine were selected.These strains were auxotrophic for guanine; and these strains were namedKMBS279 (Ppur-Δatt purR::spc purA::erm guaB(A1) ΔpupG trpC2) and KMBS280(Ppur1-Δatt purR::spc purA::erm guaB::(A1) ΔpupG trpC2), respectively.

(2) Purine Nucleoside Production by Bacterial Strains having theModified Ppur Region

Bacterial strains having the modified Ppur region (strains KMBS279 andKMBS280) and a control YMBS2 strain were uniformly spread over PS platemedium (30 g/L of soluble starch, 5 g/L of yeast extract, 5 g/L ofpolypeptone, 20 g/L of agar, adjusted to pH 7.0 with KOH) and culturedovernight at 34° C. One-eighth of the bacteria cells on the plate wereinoculated into 20 mL of fermentation medium in a 500 mL capacitySakaguchi flask. Subsequently, 50 g/L of calcium carbonate was added andthe bacteria were cultured at 34° C. with shaking. Sampling wasconducted at the time point of 96 hour after the start of culturing, andthe amounts of inosine and hydroxanthine in the culture medium weremeasured by conventional methods.

Composition of fermentation medium: Glucose 60 g/L KH₂PO₄ 1 g/L NH₄Cl 32g/L Mameno (T-N)* 1.35 g/L DL-methionine 0.3 g/L L-tryptophan 0.02 g/LAdenine 0.1 g/L Guanine 0.05 g/L MgSO₄ 0.4 g/L FeSO₄ 0.01 g/L MnSO₄ 0.01g/L GD113 0.01 mL/L (adjusted to pH 7.0 with KOH) Calcium carbonate 50g/L*Protein hydrolysis product

TABLE 3 B. subtilis strain OD562 Inosine, g/L Hypoxanthine, g/L YMBS26.5 3.08 0.32 6.5 3.15 0.25 KMBS279 4.9 3.99 0.43 5.5 4.57 0.36 KMBS2805.7 5.31 0.39 5.7 5.26 0.39

Example 5

<Construction of a Bacterium with Enhanced PRPP Synthetase Activity>

(1) The SD sequence of PRPP synthetase gene (prs) was modified in therecombinant KMBS280 strain which is derived from Bacillus subtilis (B.subtilis 168 Marburg strain; ATCC6051) and in which the purine operonrepressor gene (purR), succinyl-AMP synthase gene (purA) and purinenucleoside phosphorylase gene (pupG) had been disrupted, and a guaB (A1)mutation had been introduced into the IMP hydrogenase gene (guaB); andwhich has the modified purine operon promoter region.

(i) Amplification of Upstream Region of the SD Sequence Containing aModified SD Sequence by PCR

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimer of SEQ ID NO: 34 and the primers having the modified Ppursequence were designed:

cgcggatccAACATACACAAAGAGAAGCGAAAGC (SEQ ID NO: 34; the nucleotidesequence indicated by lower-case letters is a tag containing a BamHIsite);

<for Modification SD1>

GATTAGACATGGATAAA{overscore (CCTCCttA)}TTTAGGATTATTTTTTATGAA (SEQ ID NO:35; the nucleotides indicated by lower-case letters is a mutation point,and the sequence in the box is a modified SD sequence 1);

<for Modification SD2>

GATTAGACATGGATAAA{overscore (CCTCCtAA)}TTTAGGATTATTTTTTATGAA (SEQ ID NO:36; the nucleotide indicated by lower-case letter is a mutation point,and the sequence in the box is a modified SD sequence 2);

<for Modification SD3>

GATTAGACATGGATAAA{overscore (CCTCCGtA)}TTTAGGATTATTTTTTATGAA (SEQ ID NO:37; the nucleotide indicated by lower-case letter is a mutation point,and the sequence in the box is a modified SD sequence 3).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1.5minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe above-described primers and a template of the chromosomal DNA of B.subtilis 168 Marburg strain, to amplify a fragment containing a regionupstream of the prs start codon (about 1,040 bp) and its downstreamsequence (10 bp).

(ii) Amplification of a Region Downstream of the SD Sequence Containinga Modified SD Sequence by PCR

Based on the information from GenBank (Accession No. NC_(—)000964), PCRprimer of SEQ ID NO: 38 and the three kinds of primers having modifiedSD sequence were prepared.

cgcggatccGGTTTAGCTGAACAGATAGCTGACTGATTGC (SEQ ID NO: 38; the nucleotidesequence indicted by lower-case letters is a tag containing a BamHIsite);

<for SD1 Modification>

TTCATAAAAAATAATCCTAAATaaGGAGG|TTTATCCATGTCTAATC (SEQ ID NO: 39: thenucleotides indicated by lower-case letters are a mutation point; thesequence in the box is SD sequence 1; this primer is complementary toSEQ ID NO: 35)

<for SD2 Modification>

TTCATAAAAAATAATCCTAAA{overscore (TTaGGAGG)}TTTATCCATGTCTAATC (SEQ ID NO:40: the nucleotide indicated by lower-case letter is a mutation point;the sequence in the box is SD sequence 2; this primer is complementaryto SEQ ID NO: 36)

<for SD3 Modification>

TTCATAAAAAATAATCCTAAATaCGGAGG|TTTATCCATGTCTAATC (SEQ ID NO: 41: thenucleotide indicated by lower-case letter is a mutation point; thesequence in the box is SD sequence 3; this primer is complementary toSEQ ID NO: 37)

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1.5minute; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer)) usingthe above-described primers and a template of the chromosomal DNA of B.subtilis 168 Marburg strain, to amplify a fragment containing a regionupstream of prs start codon (36 bp) and the following downstreamsequence (about 960 bp).

(iii) Amplification of a Region Containing the Modified SD Sequence byPCR

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 2.5minutes; 30 cycles; Gene Amp PCR System Model 9600 (Perkins Elmer))using primers of SEQ ID NOS: 34 and 38 and a template of a suitablemixture of the amplified DNA fragments of the above (i) and (ii) afterpurification on a MicroSpin Column S-400 (Amersham Pharmacia Biotech),to amplify a fragment in which the SD sequence was modified to be SDsequence 1, 2, or 3, respectively (SD1, SD2, SD3).

(iv) Cloning of a Region Containing the Modified SD Sequence

The DNA fragment having a region containing the modified SD sequence(SD1, SD2, SD3) was purified and digested with BamHI, and ligated byusing T4 DNA ligase to a recombination vector pJPM1 (Mueller et al., J.Bacteriol., 1992, 174, 4361-4373) that had been digested with the sameenzyme and dephosphorylated with calf intestinal phosphatase, andthereby, the plasmids pKM196 (SD1), pKM197 (SD2), and pKM198 (SD3) wereobtained.

(v) Preparation of an Inosine-Producing Bacterial Strain in Which theModified SD Sequence is Introduced

The plasmids pKM196 (SD1), pKM197 (SD2), and pKM198 (SD3) were used totransform competent cells of the strain KMBS280 prepared as describedabove, and the colonies that were capable of growing on an LB agar platecontaining 2.5 μg/mL of chloramphenicol (single-crossover recombinant)were selected.

The obtained single-crossover recombinants were inoculated on 10 mL ofLB medium and successively subcultured for several days at 37° C. Thecolonies exhibiting chloramphenicol-sensitivity was selected on LB agarmedium. Chromosomal DNA was prepared from the chloramphenicol-sensitivecolonies. PCR was conducted in the same manner as described above usingprimers of SEQ ID NOS: 34 and 38. The DNA sequence was analyzed toidentify bacterial strains in which the SD sequence of the prs gene onthe chromosome had been replaced with the modified SD sequences by thedouble-crossover recombination. Each type of the double-crossoverrecombinants thus obtained was named: KMBS310 (SD1), KMBS318 (SDS),KMBS322 (SD3).

(2) Purine Nucleoside Production by Inosine-Producing Bacterial Strainsin Which the Modified SD Sequence is Introduced

Inosine-producing bacterial strains (strains KMBS310, KMBS318, andKMBS322) having the modified SD sequences SD1, SD2, or SD3, and acontrol KMBS280 strain were uniformly spread over a PS medium plate(soluble starch 30 g/L, yeast extract 5 g/L, polypeptone 5 g/L, agar 20g/L, adjusted to pH 7.0 with KOH) and cultured overnight at 34° C.One-eighth of the bacterial cells on the plate were inoculated on 20 mLof fermentation medium in a 500 mL capacity Sakaguchi flask.Subsequently, calcium carbonate was added at 50 g/L and the bacteriawere cultured at 34° C. with shaking. Sampling was conducted 120 hoursafter the start of culturing, and the amounts of inosine andhypoxanthine in the culture medium were measured by conventionalmethods. Glucose 60 g/L KH₂PO₄ 1 g/L NH₄Cl 32 g/L Mameno (T-N)* 1.35 g/LDL-methionine 0.3 g/L L-tryptophan 0.02 g/L Adenine 0.1 g/L Guanosine0.075 g/L MgSO₄ 0.4 g/L FeSO₄ 0.01 g/L MnSO₄ 0.01 g/L GD113 0.01 mL/L(adjusted to pH 7.0 with KOH) Calcium carbonate 50 g/L*Protein hydrolysis product

TABLE 4 B. subtilis strain OD562 Inosine (g/L) Hypoxanthine (g/L)KMBS280 4.2 3.76 0.45 4.2 3.70 0.47 KMBS310 4.4 4.88 0.55 4.4 5.07 0.53KMBS318 4.4 4.41 0.43 4.4 4.58 0.43 KMBS322 4.4 3.85 0.40 4.7 4.45 0.40

Example 6

Construction of bacterial strains having enhanced activity of an enzymeinvolved in oxidative pentosephosphate pathway

(1) Construction of a Strain having Enhanced Glucose-6-PhosphateDehydrogenase Activity

A plasmid carrying a zwf gene that encodes glucose-6-phosphatedehydrogenase was introduced into a recombinant strain which is derivedfrom Bacillus subtilis (B. subtilis 168 Marburg strain; ATCC6051) and inwhich purine operon repressor gene (purR), succinyl-AMP synthase gene(purA), and purine nucleoside phosphorylase genes (pupG and deoD) hadbeen disrupted; a guaB (A1) mutation had been introduced into IMPdehydrogenase gene; and which has the modified purine operon promoterregion and the modified SD sequence in PRPP synthetase gene (prs).

(i) Amplification of the Structural Gene and Upstream Region of the zwfGene by PCR

Based on the information from GenBank (Accession No. NC_(—)000964CAB14317. glucose-6-phospha . . . [gi:2634820]), PCR primers having thenucleotide sequences shown below were prepared:

cgcggatccGCCTCTGAAAAGAACAATCC (SEQ ID NO: 43; the nucleotide sequenceindicated by lower-case letters is a tag containing a BamHI site);

cgcggatccAAGCTCTTAAGCTTTGGGGG (SEQ ID NO: 44; the nucleotide sequenceindicated by lower-case letters is a tag containing a BamHI site).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 2minutes; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of achromosomal DNA of the B. subtilis 168 Marburg strain, to amplify afragment containing the structural gene and upstream region (about 160bp) of the zwf gene.

(ii) Cloning of the Structural Gene and Upstream Region of the zwf Gene

The DNA fragment containing the structural gene and its upstream regionof the zwf gene was purified and then digested with BamHI, and ligatedby using T4 DNA ligase to an E. coli-Bacillus shuttle vector pHY300PLK(made by Yakult Corp.) that had been digested with the same enzyme anddephosphorylated with calf intestinal phosphatase, and thereby, aplasmid containing the zwf gene was obtained.

(iii) Construction of an Inosine-Producing Bacterial Strain in Which aPlasmid Containing the zwf Gene is Introduced

The obtained plasmid or control pHY300PLK vector was used to transformcompetent cells of the inosine-producing strain KMBS321, and thecolonies that were capable of growing on LB agar plate containing 12.5μg/mL of tetracycline were selected. The strain introduced with theplasmid carrying the zwf gene was named TABS125 and the strainintroduced with pHY300PLK was named TABS100.

The KMBS321 strain was constructed as follows.

Genomic DNA was isolated from the deoD-deficient mutant strain KMBS16(purR::spc purA::erm deoD::kan: US2004-0166575A) by the method of Fouetand Sonenshein (J. Bacteriol., 1990, 172, 835-844) and the genomic DNAwas used to transform competent cells of B. subtilis 168 Marburg,prepared by the method of Dubunau and Davidoff-Abelson to obtaincolonies which were capable of growing on an LB agar plate containing 5μg/ml of kanamycin. Among the colonies which appeared, colonies whichwere not resistant to spectinomycin or erythromycin were selected, andone of such colonies was named KMBS(deoD::kan).

Genomic DNA was isolated from the KMBS(deoD::kan) by the method of Fouetand Sonenshein and the genomic DNA was used to transform competent cellsof the KMBS310 as mentioned above to obtain colonies which were capableof growing on an LB agar plate containing 20 μg/ml of guanine. Among thecolonies which appeared, a strain in which a wild-type deoD gene wasreplaced by deoD::kan and the other mutations derived from KMBS310 didnot revert to the respective wild-type genes was selected, and thestrain was named KMBS321.

An increase in glucose-6-phosphate dehydrogenase activity was confirmedas follows.

<Method of Measuring Glucose-6-Phosphatedehydrogenase (Product of thezwf Gene) Activity>

-   Reaction solution-   50 mM Tris-HCl (pH7.6)-   10 mM MgSO₄.7H₂O-   0.3 mM NADP-   4 mM glucose 6-phosphate-   Enzyme solution

The above-described reaction solution (1 mL) was prepared and thereaction was performed at 37° C. The change in concentration of theNADPH was measured based on absorption at 340 nm.

(iv) The Purine-Derived Nucleic Acid Production by the Inosine-ProducingStrains in Which the Plasmid Containing the zwf Gene is Introduced

The TABS125 strain and TABS100 strain were uniformly spread over a PSplate medium (soluble starch 30 g/L, yeast extract 5 g/L, polypeptone 5g/L, agar 20 g/L, adjusted to pH 7.0 with KOH) containing 12.5 μg/mL oftetracycline and cultured at 34° C. overnight. One-eighth of thebacterial cells on the plate were inoculated into 20 mL of fermentationmedium in a 500 mL capacity Sakaguchi flask. Subsequently, calciumcarbonate was added at 50 g/L and the bacteria were cultured at 34° C.with shaking. Sampling was conducted at 120 hours after the start ofculturing, and the amounts of inosine and hypoxanthine in the culturemedium were measured by conventional methods (Table 5). It was confirmedthat a bacterial strain with enhanced purine nucleoside-producingability was obtained.

Composition of fermentation medium: Glucose 60 g/L KH₂PO₄ 1 g/L NH₄Cl 32g/L Mameno (T-N)* 1.35 g/L Yeast extract 1 g/L DL-methionine 0.3 g/LL-tryptophan 0.02 g/L Adenine 0.1 g/L Guanosine 0.075 g/L MgSO₄ 0.4 g/LFeSO₄ 0.01 g/L MnSO₄ 0.01 g/L GD113 0.01 mL/L (adjusted to pH 7.0 withKOH) Calcium carbonate 50 g/L*Protein hydrolysis product

TABLE 5 B. subtilis strain OD610 Inosine (%)*¹ TABS100 8.4 18.73 8.719.24 TABS125 9.1 21.64 9.0 21.99*¹Ratio of the produced inosine to the amount of the consumed glucose(g/g)

(2) Construction of a Strain with Enhanced Ribose-5-Phosphate IsomeraseActivity

Expression of a gene encoding ribose-5-phosphate isomerase (ywlF gene)was enhanced in a recombinant strain which is derived from Bacillussubtilis (B. subtilis 168 Marburg strain; ATCC6051) and in which purineoperon repressor gene (purR), succinyl-AMP synthase gene (purA), andpurine nucleoside phosphorylase gene (pupG and deoD) had been disrupted;the guaB (A1) mutation had been introduced into IMP dehydrogenase gene;and which has the modified purine operon promoter region and themodified SD sequence in PRPP synthetase gene (prs).

(i) Amplification of the Structural Gene and Upstream Region of the ywlFGene by PCR

Based on the information from GenBank (Accession No. NC_(—)000964CAB15709 [gi:2636217]), PCR primers having the nucleotide sequencesshown were prepared:

cgcgaattcGTAGATAAGTTGTCAGAAAATCTGC (SEQ ID NO: 45; the nucleotidesequence indicated by lower-case letters is a tag containing an EcoRIsite);

cgcgaattcTGTTTCAACTCATTCATTAAACAGC (SEQ ID NO: 46; the nucleotidesequence indicated by lower-case letters is a tag containing an EcoRIsite).

PCR was conducted (94° C., 30 seconds; 55° C., 1 minute; 72° C., 1minute; 30 cycles; Gene Amp PCR System Model 9600 (made by PerkinsElmer)) using the above-described primers and a template of achromosomal DNA of the B. subtilis 168 Marburg strain, to amplify afragment containing the structural gene and upstream region (about 160bp) of the ywlF gene.

(ii) Cloning of the Structural Gene and Upstream Region of the ywlF Gene

The DNA fragment containing the structural gene and its upstream regionof the ywlF gene was purified and then digested with EcoRI and ligatedby using T4 DNA ligase to the E. coli-Bacillus shuttle vector pHY300PLK(made by Yakult Corp.) that had been digested with the same enzyme anddephosphorylated with calf intestinal phosphatase, and thereby a plasmidfor enhancing expression of the ywlF gene was obtained.

(iii) Preparation of an Inosine-Producing Bacterial Strain in Which thePlasmid Containing rge ywlF Gene is Introduced

The obtained plasmid or pHY300PLK vector was used to transform thecompetent cells of the inosine-producing bacterial strains KMBS321prepared as described above, and the colonies that were capable ofgrowing on an LB agar plate containing 12.5 μg/mL of tetracycline wereselected. The strain in which the plasmid carrying ywlF was introducedwas named TABS102.

An increase in ribose-5-phosphate isomerase activity was confirmed asfollows.

<Method of Detecting Ribose-5-Phosphate Isomerase (ywlF Gene Product)Activity>

-   Reaction solution-   50 mM HEPES (pH 7.5)-   0.1 M NaCl-   10 mM Ribose-5-phosphate-   Enzyme solution

The above reaction solution (mL) was prepared and the reaction wasinitiated at 37° C. The change in concentration of riboluse-5-phosphatewas measure based on absorption of 290 nm.

(iv) The Production of Purine-Derived Nucleic Acids by theInosine-Producing Bacterial Strain Introduced with the Plasmid Carryingthe ywlF Gene

The TABS102 strain and TABS100 strain were introduced was uniformlyspread over a PS medium plate (soluble starch 30 g/L, yeast extract 5g/L, polypeptone 5 g/L, agar 20 g/L, adjusted to pH 7.0 with KOH)containing 12.5 μg/mL of tetracycline and the bacteria were cultured at34° C. overnight. One-eighth of the bacterial cells on the plate wereinoculated into 20 mL of fermentation medium in a 500 mL capacitySakaguchi flask. Subsequently, calcium carbonate was added at 50 g/L andthe bacteria were cultured at 34° C. with shaking. Sampling wasconducted at 120 hours after the start of culturing, and the amounts ofinosine and hypoxanthine in the culture medium were measured byconventional methods (Table 6). It was found that the strain withenhanced ribose-5-phosphate isomerase activity produced inosine moreefficiently than the unmodified strain.

Composition of the fermentation medium: Glucose 60 g/L KH₂PO₄ 1 g/LNH₄Cl 32 g/L Mameno (T-N)* 1.35 g/L Yeast extract 1 g/L DL-methionine0.3 g/L L-tryptophan 0.02 g/L Adenine 0.1 g/L Guanosine 0.075 g/L MgSO₄0.4 g/L FeSO₄ 0.01 g/L MnSO₄ 0.01 g/L GD113 0.01 mL/L (adjusted to pH7.0 with KOH) Calcium carbonate 50 g/L*Protein hydrolysis product

TABLE 6 Residual B. subtilis strain OD610 glucose (g/L) Inosine (g/L)Inosine (%)*¹ TABS100 8.4 0 5.77 18.73 TABS102 9.8 1.3 5.96 20.21*¹Ratio of the produced inosine to the amount of the consumed glucose(g/g)

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

1. A Bacillus bacterium having an ability to produce a purine-derivedsubstance, wherein the bacterium is modified to enhance an activity ofan enzyme of the oxidative pentosephosphate pathway.
 2. The Bacillusbacterium according to claim 1, wherein said purine-derived substance isa purine nucleoside selected from the group consisting of inosine,xanthosine, guanosine, and adenosine.
 3. The Bacillus bacteriumaccording to claim 1, wherein said purine-derived substance is a purinenucleotide selected from the group consisting of inosinic acid,xanthylic acid, guanylic acid, and adenylic acid.
 4. The Bacillusbacterium according to claim 1, wherein said enzyme isglucose-6-phosphate dehydrogenase or ribose-5-phosphate isomerase. 5.The Bacillus bacterium according to claim 1, wherein the activity ofsaid enzyme is enhanced by increasing the copy number of a gene encodingthe enzyme or modifying an expression control sequence of the gene. 6.The Bacillus bacterium according to claim 5, wherein said enzyme isglucose-6-phosphate dehydrogenase which is selected from the groupconsisting of: (A) a protein comprising the amino acid sequence of SEQID NO: 48; and (B) a protein comprising the amino acid sequence of SEQID NO: 48 wherein one or several amino acids are substituted, deleted,inserted, added, or inverted, and said protein has glucose-6-phosphatedehydrogenase activity.
 7. The Bacillus bacterium according to claim 5,wherein said enzyme is ribose-5-phosphate isomerase which is selectedfrom the group consisting of: (A) a protein comprising the amino acidsequence of SEQ ID NO: 50; and (B) a protein comprising the amino acidsequence of SEQ ID NO: 50 wherein one or several amino acids aresubstituted, deleted, inserted, added, or inverted, and said protein hasribose-5-phosphate isomerase activity.
 8. The Bacillus bacteriumaccording to claim 6, wherein 1-20 amino acids are substituted, deleted,inserted, added, or inverted.
 9. The Bacillus bacterium according toclaim 5, wherein said enzyme is glucose-6-phosphate dehydrogenase andthe gene encoding said enzyme is selected from the group consisting of:(A) a DNA comprising the nucleotide sequence of SEQ ID NO: 47; and (B) aDNA that is able to hybridize with a nucleotide sequence complementaryto the nucleotide sequence of SEQ ID NO: 47, or a probe prepared fromthe nucleotide sequence under stringent conditions, and wherein said DNAencodes a protein having glucose-6-phosphate dehydrogenase activity. 10.The Bacillus bacterium according to claim 5, wherein said enzyme isribose-5-phosphate isomerase and the gene encoding said enzyme isselected from the group consisting of: (A) a DNA comprising thenucleotide sequence of SEQ ID NO: 49; and (B) a DNA that is able tohybridize with a nucleotide sequence complementary to the nucleotidesequence of SEQ ID NO: 49, or a probe prepared from the nucleotidesequence under stringent conditions, and wherein said DNA encodes aprotein having ribose-5-phosphate isomerase activity.
 11. The Bacillusbacterium according to claim 1, wherein the bacterium is furthermodified to enhance phosphoribosylpyrophosphate synthetase activity. 12.The Bacillus bacterium according to claim 1, wherein the bacterium isfurther modified to enhance the expression of purine operon.
 13. TheBacillus bacterium according to claim 12, wherein the expression of thepurine operon is enhanced by disrupting a purR gene that encodes apurine operon repressor or deleting a portion of an attenuator region ofthe purine operon.
 14. The Bacillus bacterium according to claim 1,wherein the bacterium is further modified to reduce the activity ofpurine nucleoside phosphorylase.
 15. A method for producing apurine-derived substance comprising: culturing a Bacillus bacterium in amedium; and collecting said purine-derived substance, wherein saidBacillus bacterium is able to produce a purine-derived substance and hasbeen modified to enhance an activity of glucose-6-phosphatedehydrogenase, wherein said glucose-6-phosphate dehydrogenase isselected from the group consisting of: (A) a protein comprising theamino acid sequence of SEQ ID No. 48, and (B) a protein comprising theamino acid sequence of SEQ ID No. 48 wherein one or several amino acidsare substutited, deleted, inserted, added, or inverted, and said proteinhas glucose-6-phosphate dehydrogenase activity.
 16. The method accordingto claim 15, wherein said purine-derived substance is a purinenucleoside or purine nucleotide.
 17. The method according to claim 16,wherein said purine-derived substance is selected from the groupconsisting of inosine, xanthosine, guanosine, and adenosine.
 18. Themethod according to claim 16, wherein said purine-derived substance isselected from the group consisting of inosinic acid, xanthylic acid,guanylic acid, and adenylic acid.
 19. A method for producing a purinenucleotide comprising: producing a purine nucleoside by the methodaccording to claim 17; and reacting the purine nucleoside with amicroorganism which has an ability to produce a nucleoside-5′-phosphateester, or with an acid phosphatase, in the presence of a phosphate donorselected from the group consisting of phosphoric acid, phenyl phosphate,and carbamyl phosphate; and collecting the purine nucleotide.
 20. Themethod according to claim 15, wherein 1-20 amino acids are substituted,deleted, inserted, added, or inverted.
 21. The method according to claim15, wherein the gene encoding said protein is selected from the groupconsisting of: (A) a DNA comprising the nucleotide sequence of SEQ IDNO: 47; and (B) a DNA that is able to hybridize with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID NO: 47, or aprobe prepared from the nucleotide sequence under stringent conditions,and wherein said DNA encodes a protein having glucose-6-phosphatedehydrogenase activity.
 22. The method according to claim 15, whereinthe bacterium is further modified to enhance phosphoribosylpyrophosphatesynthetase activity.
 23. The method according to claim 15, wherein thebacterium is further modified to enhance the expression of purineoperon.
 24. The method according to claim 23, wherein the expression ofthe purine operon is enhanced by disrupting a purR gene that encodes apurine operon repressor or deleting a portion of an attenuator region ofthe purine operon.
 25. The method according to claim 15, wherein thebacterium is further modified to reduce the activity of purinenucleoside phosphorylase.
 26. The method according to claim 15, whereinthe activity of said protein is enhanced by increasing the copy numberof a gene encoding the protein or modifying an expression controlsequence of the gene.